Dimeric Small Molecule Potentiators of Apoptosis

ABSTRACT

Caspase activity and apoptosis are promoted using active, dimeric Smac peptide mimetics of the general formula M1-L-M2, wherein moieties M1 and M2 are monomeric Smac mimetics and L is a covalent linker. Target cancerous or inflammatory cells are contacted with an effective amount of an active, dimeric Smac mimetic, and a resultant increase in apoptosis of the target cells is detected. The contacting step may be effected by administering to a pharmaceutical composition comprising a therapeutically effective amount of the dimeric mimetic, wherein the individual may be subject to concurrent or antecedent radiation or chemotherapy for treatment of a neoproliferative pathology.

This application is a continuation of U.S. Ser No. 11/070,733, filedMar. 1, 2005 (U.S. Pat. No. 7,309,792), which is a continuation of U.S.Ser. No. 60/549,520 filed Mar. 1, 2004.

This work was supported by National Institute of Health Grant No. P01CA9547101. The U.S. government may have rights in any patent issuing onthis application.

INTRODUCTION

1. Field of the Invention

The field of the invention is dimeric small molecule potentiators ofapoptosis

2. Background of the Invention

Apoptosis plays a central role in the development and homeostasis of allmulti-cellular organisms. Abnormal inhibition of apoptosis is a hallmarkof cancer and autoimmune diseases, whereas excessive activation of celldeath is implicated in neuro-degenerative disorders such as Alzheimer'sdisease. Pro-apoptotic chemotherapeutic drugs provide a recent approachto overcoming the clinical problem of drug resistance; see, e.g. Makinet al., Cell Tissue Res 2000 July; 301(1):143-52 (“Apoptosis and cancerchemotherapy”).

The mechanism of apoptosis is conserved across species and executed witha cascade of sequential activation of proteases called caspases. Onceactivated, these caspases are responsible for proteolytic cleavage of abroad spectrum of cellular targets that ultimately lead to cell death.IAPs (inhibitor-of-apoptosis proteins) regulate apoptosis by inhibitingcaspases; and a protein called Smac (second mitochondria-derivedactivator of caspases) binds to and inhibits IAPs, and thereby promotescaspase activation. N-terminal Smac-derived peptides and mimetics havebeen shown to similarly inhibit IAPs, and promote caspase activation.IAPs are components of TNFR (tumor necrosis factor receptor), so IAPinhibitors can divert TNFR signalling from an NfkB-mediatedpro-inflammatory signal, to an anti-inflammatory apoptotic signal.

RELEVANT LITERATURE

-   Liu et al, Structural basis for binding of Smac/DIABLO to the XIAP    BIR3 domain; Nature 2000 Dec. 21-28; 408(6815):1004-8.-   Wu et al., Structural basis of IAP recognition by Smac/DIABLO;    Nature 2000 Dec. 21-28; 408(6815): 1008-12.-   Fesik, et al., Peptides derived from smac (DIABLO) and methods of    using them to screen for apoptosis modulating compounds; WO    2002030959.-   McLendon, et al., IAP binding peptides and assays for identifying    compounds that bind IAP; WO 2002096930.-   Shi, Compositions and methods for regulating apoptosis; WO    2002026775.-   Debatin, et al., Smac-peptides as therapeutics against cancer and    autoimmune diseases by sensitizing for TRAIL- or anticancer    drug-induced apoptosis; WO 2003086470.-   Alnemri, Conserved sequence of XIAP-binding motif in human caspase-9    and Smac/DIABLO and therapeutic uses for screening modulators of    apoptosis; WO 2003010184.-   Arnt et al., Synthetic Smac/DIABLO peptides enhance the effects of    chemotherapeutic agents by binding XIAP and cIAP1 in situ; J Biol    Chem. 2002 Nov. 15; 277(46):44236-43. Epub 2002 Sep. 5.-   IAP binding peptide or polypeptide and methods of using the same; US    Pat Publ No. 20020132786.-   US Pat Publ No. 20020160975, Conserved XIAP-interaction motif in    caspase-9 and Smac/DIABLO for mediating apoptosis.-   US Pat Publ No. 20020177557, Compositions and method for regulating    apoptosis.-   U.S. Pat. No. 6,608,026, Wang, et al., Apoptotic Compounds;-   Li et al., Targeting and amplification of immune killing of tumor    cells by pro-Smac, Int J Cancer. 2004 Mar. 10; 109(1):85-94.

SUMMARY OF THE INVENTION

We have serendipitously discovered that dimeric versions ofpro-apoptotic Smac peptides and peptide mimetics provide vastly improvedreagents over the prior art monomers. These dimers similarly bindcytosolic IAPs and relieve their inhibition of caspases; however, theydo so with exceptional potency. We believe they bind to adjacent repeatsof the BIR (baculoviral inhibitory repeat) domain within IAP proteins,and that this bipartite recognition is crucial to their effectiveness:the corresponding monomers typically have much less activity in cellextracts (e.g. ˜10000 times less active). In fact, our data indicatethat dimers function catalytically, shunting uncomplexed IAP to theubiquitin/proteasome degradation pathway. The dimers synergize withTRAIL (TNF-related apoptosis inducing ligand) to induce apoptosis inglioblastoma cell culture, typically at picomolar concentrations. Thecompounds provide new adjuvant chemotherapeutics for cancers,particularly those that resist programmed cell death by over-expressingIAP proteins.

As a preferred embodiment, we developed novel series of dimericmolecules that also mimic the endogenous function of Smac, and providevastly enhanced activity over prior Smac mimetics. The compounds arestable, protease resistant, and freely membrane permeant; by themselves,the molecules are not cytotoxic. No similar compounds have beendescribed. Though others have attempted to use Smac-derived peptides andpeptide-carrier constructs for similar purposes, these prior artmaterials are severely limited by their physicochemical properties andtheir potency is orders of magnitude less than that disclosed here.

Accordingly, the invention provides methods and compositions forenhancing apoptosis of pathogenic cells using pro-apoptotic dimeric Smacpeptide mimetics. The general method comprises the step of contactingthe cells with an effective amount of an active, dimeric Smac mimetic,typically followed by the step detecting, directly, indirectly orinferentially a resultant increase in apoptosis of the target cells.Dimer activity may be determined by IAP binding, procaspase-3 activationor promotion of apoptosis, etc.

In preferred embodiments, the cells are in situ in an individual and thecontacting step is effected by administering to the individual apharmaceutical composition comprising a therapeutically effective amountof the mimetic, wherein the individual may be subject to concurrent orantecedent radiation or chemotherapy for treatment of a neoproliferativepathology. In particular embodiments, the pathogenic cells are of atumor, such as a tumor selected from the group consisting ofglyoblastoma, astrocytoma, breast cancer, prostate cancer, lung cancer,pancreatic cancer, gastric cancer, colon cancer, ovarian cancer, renalcancer, hepatoma, melanoma, lymphoma, and sarcoma. In additionalembodiments, the target cells are pro-inflammatory cells or cells oftissue subject to pathogenic inflammation and/or autoimmunity. A widevariety of diseased provide target pathogenic inflammation, includingrheumatoid arthritis, diabetes, asthma, lupus, inflammatory boweldisease (Crohn's disease and related conditions), multiple sclerosis,chronic obstructive pulmonary disease, inflammatory bowel and pelvicdiseases, allergic rhinitis (hay fever), cardiovascular disease, etc.

The subject compositions encompass pharmaceutical compositionscomprising a therapeutically effective amount of an active, dimeric Smacmimetic in dosage form and a pharmaceutically acceptable carrier. Insome embodiments, such compositions further comprise an additionaltherapeutic agent, such as an anti-neoproliferative chemotherapeuticagent, other than the mimetic.

The invention also provides methods for making and screening for activedimeric Smac peptide mimetics. For example, one general assay comprisesthe steps of generating a dimeric Smac peptide mimetic from a monomericmimetic, and detecting enhanced activity of the resultant dimer over themonomeric precursor, e.g. as determined by IAP binding, procaspase-3activation or promotion of apoptosis.

The subject mimetics encompass a wide variety of active, dimeric Smacmimetics of the general formula M1-L-M2, wherein moieties M1 and M2 aremonomeric Smac mimetics and L is a linker covalently linking M1 and M2in the active dimer. M1 and M2 each encompass monomeric Smac mimetics,particularly pro-apoptotic, particularly AVP-type and AV peptoidmimetics; see e.g. WO 2002030959; WO 2002096930; WO 2002026775; WO2003086470; WO 2003010184; US Pat Publ Nos. 20020177557, 20020132786,20020160975; U.S. Pat. No. 6,608,026; etc, and include the varied anddiverse monomeric mimetic structures disclosed or referenced herein.

The linker L serves to covalently couple M1 and M2 in a dimericstructure that provides enhanced pro-apoptotic activity over theuncoupled monomers, and is otherwise compatible with the disclosed usesof the dimers (e.g. physiological compatibility and stability). A widevariety of linkers may be used, and particular linkers are readilyassayed empirically. Generally, L is a contiguous chain of between 2 and200 atoms, preferably between 4 and 100 atoms, more preferably between 4and 25 atoms, and a MW between 20 and 2 K D, preferably between 40 and 1K D, more preferably between 56 and 1 K D. L maybe bisymmmetrical, ornonsymmetrical, and may link different, isometric or identical M1 andM2, typically has spans between about 3 and 3 K A, preferably betweenabout 6 and 2000, more preferably between about 12 and 1000 A, etc.Exemplary, nonlimiting suitable linkers are further described below.

In a particular embodiment, the invention provides a dimeric compound offormula II,

wherein:

R1 and R1′ are selected from hydrogen, optionally substituted methyl,and hydroxyl; R2 and R2′ are selected from optionally substituted methyland optionally substituted ethyl; R3 and R3′ are selected from CH2, NH,O and S; R4 and R4′ are selected from CH and N; R5-R8, and R5′-R8′ areselected from hydrogen, optionally hetero-, optionally substitutedalkyl, optionally hetero-, optionally substituted alkenyl, optionallyhetero-, optionally substituted alkynyl, optionally hetero-, optionallysubstituted aryl; and L is a linker covalently linking R2, R5, R6 or R7,with R2′, R5′, R6′ or R7′,

or a pharmaceutically-acceptable salt thereof.

Various particular embodiments include all combinations wherein:

R1 and R1′ are selected from hydrogen and methyl; R2 and R2′ areselected from methyl and ethyl; R3 and R3′ are NH; R4 and R4′ are CH; R5and R5′ are C1-C3 alkyl; and

R6/R6′ and R7/R7′, or R7/R7′ and R8/R8′ are connected in a 5- to8-membered ring; more particularly wherein R1 and R1′ are selected fromhydrogen and methyl, R2 and R2′ are selected from methyl and ethyl, R3and R3′ are NH, R4 is CH, and R5 and R5′ are C1-C3 alkyl, and Lcovalently links R5, R6 or R7, with R5′, R6′ or R7′,

More particular embodiments include all combinations wherein:

R1/R1′ and R2/R2′ are connected to form a 4-membered ring (azetidine);R7 and R8 are connected in a 5- or 6-membered ring; R6 and R7 areconnected in a 5- or 6-membered ring, particularly wherein R6 and R7 areconnected in a 5-membered ring, and L covalently links the ring withR2′, R5′, R6′ or R7′; and R8 comprises a 5- or 6-membered ring,particularly wherein R8 comprises a 5-membered ring, comprising at leastone heteroatom, at least one substitution, and at least oneunsaturation.

In particular embodiments, the L is a contiguous chain of between 4 and100 atoms, and between 40 and 1 kD, and is an optionally hetero-,optionally substituted dialkynyl radical. In particular embodiments, thelinker is bisymmetrical about the linker; in particular embodiments, thedimer itself is bisymmetrical.

The invention also provides pharmaceutical compositions comprising thesubject compounds and a pharmaceutically acceptable excipient,particularly such compositions comprising a unit dosage of the subjectcompounds, particularly such compositions copackaged with instructionsdescribing use of the composition to treat a disease associated withundesirably high IAP activity, and/or undesirably low caspase orapoptotic activity, particularly as found in many tumors andinflammation.

Accordingly, the invention provides methods of treating a diseaseassociated with undesirable caspase activity, the method comprising thestep of administering an effective dosage of the subject compounds andcompositions, which may be followed by the step of detecting a resultantdecrease in pathology associated with the disease, and which may beprefaced by the step of diagnosis such disease and/or prescribing suchcomposition. Applicable disease include tumors and inflammation.

The invention also provides methods of inhibiting a caspase, the methodcomprising the step of contacting a composition comprising a caspasewith an effective amount of the subject compounds and compositions,which may be followed by the step of detecting a resultant change incaspase activity.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

The following descriptions of particular embodiments and examples areoffered by way of illustration and not by way of limitation. Unlesscontraindicated or noted otherwise, in these descriptions and throughoutthis specification, the terms “a” and “an” mean one or more, the term“or” means and/or and polynucleotide sequences are understood toencompass opposite strands as well as alternative backbones describedherein.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which is fully saturated,having the number of carbon atoms designated (i.e. C1-C8 means one toeight carbons). Examples of alkyl groups include methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like.

The term “alkenyl”, by itself or as part of another substituent, means astraight or branched chain, or cyclic hydrocarbon radical, orcombination thereof, which may be mono- or polyunsaturated, having thenumber of carbon atoms designated (i.e. C2-C8 means two to eightcarbons) and one or more double bonds. Examples of alkenyl groupsinclude vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl) and higher homologs and isomersthereof.

The term “alkynyl”, by itself or as part of another substituent, means astraight or branched chain hydrocarbon radical, or combination thereof,which may be mono- or polyunsaturated, having the number of carbon atomsdesignated (i.e. C2-C8 means two to eight carbons) and one or moretriple bonds. Examples of alkynyl groups include ethynyl, 1- and3-propynyl, 3-butynyl and higher homologs and isomers thereof.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from alkyl, as exemplified by—CH2-CH2-CH2-CH2-. Typically, an alkyl (or alkylene) group will havefrom 1 to 24 carbon atoms, with those groups having 10 or fewer carbonatoms being preferred in the present invention. A “lower alkyl” or“lower alkylene” is a shorter chain alkyl or alkylene group, generallyhaving eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and from one to three heteroatoms selectedfrom the group consisting of O, N, Si and S, wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S may be placed atany interior position of the heteroalkyl group. The heteroatom Si may beplaced at any position of the heteroalkyl group, including the positionat which the alkyl group is attached to the remainder of the molecule.Examples include —CH2-CH2-O—CH3, —CH2-CH2-NH—CH3, —CH2-CH2-N(CH3)-CH3,—CH2-S—CH2-CH3, —CH2-CH2, —S(O)—CH3, —CH2-CH2-S(O)2-CH3, —CH═CH—O—CH3,—Si(CH3)3, —CH2-CH═N—OCH3, and —CH═CH—N(CH3)-CH3. Up to two heteroatomsmay be consecutive, such as, for example, —CH2-NH—OCH3 and—CH2-O—Si(CH3)3.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified by —CH2-CH2-S—CH2-CH2- and —CH2-S—CH2-CH2-NH—CH2-. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Accordingly, acycloalkyl group has the number of carbon atoms designated (i.e., C3-C8means three to eight carbons) and may also have one or two double bonds.A heterocycloalkyl group consists of the number of carbon atomsdesignated and from one to three heteroatoms selected from the groupconsisting of O, N, Si and S, and wherein the nitrogen and sulfur atomsmay optionally be oxidized and the nitrogen heteroatom may optionally bequaternized. Additionally, for heterocycloalkyl, a heteroatom can occupythe position at which the heterocycle is attached to the remainder ofthe molecule. Examples of cycloalkyl include cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include 1-(1,2,5,6-tetrahydropyrid-yl), 1-piperidinyl,2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl,tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” and “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include alkyl substituted with halogen atoms, which can be thesame or different, in a number ranging from one to (2m′+1), where m′ isthe total number of carbon atoms in the alkyl group. For example, theterm “halo(C1-C4)alkyl” is mean to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. Thus,the term “haloalkyl” includes monohaloalkyl (alkyl substituted with onehalogen atom) and polyhaloalkyl (alkyl substituted with halogen atoms ina number ranging from two to (2m′+1) halogen atoms, where m′ is thetotal number of carbon atoms in the alkyl group). The term“perhaloalkyl” means, unless otherwise stated, alkyl substituted with(2m′+1) halogen atoms, where m′ is the total number of carbon atoms inthe alkyl group. For example the term “perhalo(C1-C4)alkyl” is meant toinclude trifluoromethyl, pentachloroethyl,1,1,1-trifluoro-2-bromo-2-chloroethyl and the like.

The term “acyl” refers to those groups derived from an organic acid byremoval of the hydroxy portion of the acid. Accordingly, acyl is meantto include, for example, acetyl, propionyl, butyryl, decanoyl, pivaloyl,benzoyl and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,typically aromatic, hydrocarbon substituent which can be a single ringor multiple rings (up to three rings) which are fused together or linkedcovalently. Non-limiting examples of aryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl and 1,2,3,4-tetrahydronaphthalene.

The term heteroaryl,” refers to aryl groups (or rings) that contain fromzero to four heteroatoms selected from N, O, and S, wherein the nitrogenand sulfur atoms are optionally oxidized and the nitrogen heteroatom areoptionally quaternized. A heteroaryl group can be attached to theremainder of the molecule through a heteroatom. Non-limiting examples ofheteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyland 6-quinolyl.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) is meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (as well as thosegroups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl andheterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′, halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO2R′, —CONR′R″, —OC(O)NR′R″, —NR″ C(O)R′, —NR′—C(O)NR″R′″,—NR′—SO2NR′″, —NR″CO2R′, —NH—C(NH2)=NH, —NR′C(NH2)=NH, —NH—C(NH2)=NR′,—S(O)R′, —SO2R′, —SO2NR′R″, —NR″SO2R, —CN and —NO2, in a number rangingfrom zero to three, with those groups having zero, one or twosubstituents being particularly preferred. R′, R″ and R′″ eachindependently refer to hydrogen, unsubstituted (C1-C8)alkyl andheteroalkyl, unsubstituted aryl, aryl substituted with one to threehalogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, oraryl-(C1-C4)alkyl groups. When R′ and R″ are attached to the samenitrogen atom, they can be combined with the nitrogen atom to form a 5-,6- or 7-membered ring. For example, —NR′R″ is meant to include1-pyrrolidinyl and 4-morpholinyl. Typically, an alkyl or heteroalkylgroup will have from zero to three substituents, with those groupshaving two or fewer substituents being preferred in the presentinvention. More preferably, an alkyl or heteroalkyl radical will beunsubstituted or monosubstituted. Most preferably, an alkyl orheteroalkyl radical will be unsubstituted. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups such as trihaloalkyl (e.g., —CF3 and—CH2CF3).

Preferred substituents for the alkyl and heteroalkyl radicals areselected from: —OR′, ═O, —NR′R″, —SR′, halogen, —SiR′R″R′″, —OC(O)R′,—C(O)R′, —CO2R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR″CO2R′,—NR′—SO2NR″R′″, —S(O)R′, —SO2R′, —SO2NR′R″, —NR″SO2R, —CN and —NO2,where R′ and R″ are as defined above. Further preferred substituents areselected from: —OR′, ═O, —NR′R″, halogen, —OC(O)R′, —CO2R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR″CO2R′, —NR′—SO2NR″R′″, —SO2R′, —SO2NR′R″,—NR″SO2R, —CN and —NO2.

Similarly, substituents for the aryl and heteroaryl groups are variedand selected from: halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN,—NO2, —CO2R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″CO2R′,—NR′—C(O)NR″R′″, —NR′—SO2NR″R′″, —NH—C(NH2)=NH, —NR′C(NH2)=NH,—NH—C(NH2)=NR′, —S(O)R′, —SO2R′, —SO2NR′R″, —NR″SO2R, —N3, —CH(Ph)2,perfluoro(C1-C4)alko-xy and perfluoro(C1-C4)alkyl, in a number rangingfrom zero to the total number of open valences on the aromatic ringsystem; and where R′, R″ and R′″ are independently selected fromhydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl andheteroaryl, (unsubstituted aryl)-(C1-C4)alkyl and (unsubstitutedaryl)oxy-(C1-C4)alkyl. When the aryl group is1,2,3,4-tetrahydronaphthalene, it may be substituted with a substitutedor unsubstituted (C3-C7)spirocycloalkyl group. The(C3-C7)spirocycloalkyl group may be substituted in the same manner asdefined herein for “cycloalkyl”. Typically, an aryl or heteroaryl groupwill have from zero to three substituents, with those groups having twoor fewer substituents being preferred in the present invention. In oneembodiment of the invention, an aryl or heteroaryl group will beunsubstituted or monosubstituted. In another embodiment, an aryl orheteroaryl group will be unsubstituted.

Preferred substituents for aryl and heteroaryl groups are selected from:halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN, —NO2, —CO2R′, —CONR′R″,—C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —S(O)R′, —SO2R′, —SO2NR′R″, —NR″SO2R,—N3, —CH(Ph)2, perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl, whereR′ and R″ are as defined above. Further preferred substituents areselected from: halogen, —OR′, —OC(O)R′, —NR′R″, —R′, —CN, —NO2, —CO2R′,—CONR′R″, —NR″C(O)R′, —SO2R′, —SO2NR′R″, —NR″SO2R,perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl.

The substituent —CO2H, as used herein, includes bioisostericreplacements therefor; see, e.g., The Practice of Medicinal Chemistry;Wermuth, C. G., Ed.; Academic Press: New York, 1996; p. 203.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CH2)q-U—, wherein T and U are independently —NH—, —O—, —CH2- ora single bond, and q is an integer of from 0 to 2. Alternatively, two ofthe substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula -A-(CH2)r-B—,wherein A and B are independently —CH2-, —O—, —NH—, —S—, —S(O)—,—S(O)2-, —S(O)2NR′— or a single bond, and r is an integer of from 1 to3. One of the single bonds of the new ring so formed may optionally bereplaced with a double bond. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula —(CH2)s-X—(CH2)t-, where s and t areindependently integers of from 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—,—S(O)2-, or —S(O)₂NR′—. The substituent R′ in —NR′— and —S(O)2NR′— isselected from hydrogen or unsubstituted (C1-C6)alkyl.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic,succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al. (1977) J. Pharm. Sci. 66: 1-19).Certain specific compounds of the present invention contain both basicand acidic functionalities that allow the compounds to be converted intoeither base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent. Prodrugs are oftenuseful because, in some situations, they may be easier to administerthan the parent drug. They may, for instance, be more bioavailable byoral administration than the parent drug. The prodrug may also haveimproved solubility in pharmacological compositions over the parentdrug. A wide variety of prodrug derivatives are known in the art, suchas those that rely on hydrolytic cleavage or oxidative activation of theprodrug. An example, without limitation, of a prodrug would be acompound of the present invention which is administered as an ester (the“prodrug”), but then is metabolically hydrolyzed to the carboxylic acid,the active entity. Additional examples include peptidyl derivatives of acompound of the invention.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are all intended to beencompassed within the scope of the present invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, animal or human that is being sought by theresearcher, veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the symptoms of the conditionsor disorder being treated. The therapeutically effective amount willvary depending on the compound, the disease and its severity and theage, weight, etc., of the mammal to be treated.

In a particular embodiment, the invention provides a dimeric compoundM1-L-M2, wherein M1 and M2 are independently a moiety of formula I, andcovalently bound through the molecular linker L through R2, R5, R6 orR7:

This particular embodiment may also be characterized as a dimericcompound of formula II,

including all combinations wherein:

R1 and R1′ are selected from hydrogen, optionally substituted methyl,and hydroxyl; R2 and R2′ are selected from optionally substituted methyland optionally substituted ethyl; R3 and R3′ are selected from CH2, NH,O and S; R4 and R4′ are selected from CH and N; R5-R8, and R5′-R8′ areselected from hydrogen, optionally hetero-, optionally substitutedalkyl, optionally hetero-, optionally substituted alkenyl, optionallyhetero-, optionally substituted alkynyl, optionally hetero-, optionallysubstituted aryl; and L is a linker covalently linking R2, R5, R6 or R7,with R2′, R5′, R6′ or R7′,

or a pharmaceutically-acceptable salt thereof.

One or more corresponding R and R′ groups may be the same or different.Various particular embodiments include all combinations wherein:

R1 and R1′ are selected from hydrogen and methyl; R2 and R2′ areselected from ethyl, and preferably methyl; R3 and R3′ are selected fromCH2, O, and preferably NH; R4 and R4′ are CH; R5 and R5′ are C1-C3alkyl; and R6/R6′ and R7/R7′, or R7/R7′ and R8/R8′ are connected in a 5-to 8-membered ring.

In more particular embodiments, R1 and R1′ are selected from hydrogenand methyl, R2 and R2′ are selected from methyl and ethyl, R3 and R3′are NH, R4 is CH, and R5 and R5′ are C1-C3 alkyl, and L covalently linksR5, R6 or R7, with R5′, R6′ or R7′.

Particular embodiments include all combinations wherein:

R1/R1′ and R2/R2′ are connected to form a 4-membered ring (azetidine);R7 and R8 are connected in a 5- or 6-membered ring; R6 and R7 areconnected in a 5- or 6-membered ring, particularly wherein R6 and R7 areconnected in a 5-membered ring, and L covalently links the ring withR2′, R5′, R6′ or R7′; and R8 comprises a 5- or 6-membered ring,particularly wherein R8 comprises a 5-membered ring, comprising at leastone heteroatom, at least one substitution, and at least oneunsaturation.

In particular embodiments, the L is a contiguous chain of between 4 and100 atoms, and between 40 and 1 kD, and is an optionally hetero-,optionally substituted dialkynyl radical. In particular embodiments, thelinker is bisymmetrical; and in more particular embodiments, the dimeritself is symmetrical, i.e. the linker is bisymmetrical, and M1 and M2are isomers (the corresponding R and R′ groups are the same).

In a particular embodiment, the L is substituted with or is a contiguouschain incorporating an aminated moiety, particularly an amino acid suchas lysine or arginine or its biostere, wherein the amine can beprotonated or alykylated to create a positive charge, and to make thecompound a triamine with three potential points of contact with a BIRdomain (see, e.g. Exemplary Dimers.

These and other embodiments of the invention are found and/orexemplified in the sections entitled Exemplary Dimers, ExemplaryMonomers, and Experimental Procedures.

The invention also provides pharmaceutical compositions comprising thesubject compounds and a pharmaceutically acceptable excipient,particularly such compositions comprising a unit dosage of the subjectcompounds, particularly such compositions copackaged with instructionsdescribing use of the composition to treat a disease associated withundesirably high IAP activity, and/or undesirably low caspase orapoptotic activity, particularly as found in many tumors andinflammation.

Accordingly, the invention provides methods of treating a diseaseassociated with undesirable caspase activity, the method comprising thestep of administering an effective dosage of the subject compounds andcompositions, which may be followed by the step of detecting a resultantdecrease in pathology associated with the disease, and which may beprefaced by the step of diagnosis such disease and/or prescribing suchcomposition. Applicable disease include tumors and inflammation.

The invention also provides methods of inhibiting a caspase, the methodcomprising the step of contacting a composition comprising a caspasewith an effective amount of the subject compounds and compositions,which may be followed by the step of detecting a resultant change incaspase activity.

The exemplified mimetics are nonlimiting. Peptide mimetic chemistry is awell-established art wherein skilled practitioners can readily generatea wide variety of mimics using conventional chemistry (see, e.g. Liao etal. (1998) J. Med. Chem 41, 4767-4776; Andrade-Gordon et al. (1999) PNASUSA 96, 12257-12262; Boatman et al. (1999) J. Med. Chem. 42, 1367-1375;Kasher et al. (1999) J. Mol. Biol 292, 421-429; U.S. Pat. No. 5,981,467;etc.) and these other strategies are applicable here, so long as theresultant mimetics are screened for and demonstrated to provide therequisite activity as described herein.

Synthetic methods for producing mimetics are well-known in the art. Somegeneral means for the production of peptides, analogs or derivatives areoutlined in Chemistry and Biochemistry of Amino Acids, Peptides andProteins, A Survey of Recent Developments, Weinstein, B. ed., MarcellDekker, Inc., publ. New York (1983). A wide variety of well-establishedtechniques are available for synthesizing peptide mimetics, see, e.g.submonomer method of R. Zuckermann et al., J. Am. Chem. Soc. (1992)0114: 10646-7. Synthesis by solid phase techniques of heterocyclicorganic compounds in which N-substituted glycine monomer units forms abackbone is described in U.S. Pat. No. 5,958,792, wherein combinatoriallibraries of mixtures of such heterocyclic organic compounds can then beassayed for the ability to inhibit IAP as described below. Highlysubstituted cyclic structures can be synthesized on a solid support bycombining the submonomer method with powerful solution phase chemistry.Cyclic compounds containing one, two, three or more fused rings areformed by the submonomer method by first synthesizing a linear backbonefollowed by subsequent intramolecular or intermolecular cyclization,also as described in U.S. Pat. No. 5,958,792. General preparativeprotocols for exemplary mimetic classes are as follows:

Preparation of α-Polyesters Using Chiral α-Hydroxy Acids As BuildingBlocks. The α-polyester structures can be prepared by using chemicalsynthesis technology known to those skilled in the art. For details ofthe reaction, see Brewster, P., et al., Nature, (1990) 166:179. Analternative method for producing similar structures is disclosed inChan, P. C., and Chong, J. M., Tetrahedron Lett. (1990)1985. Further,various publications cited within the Chan et al. publication describetechniques for synthesizing chiral α-hydroxy acids.

Preparation of Polythioamides Using Chiral .alpha.-Amino Acids AsBuilding Blocks. Polythioamide structures can be synthesized usingtechniques such as those described in Clausen, K., et al., J. Chem. Soc.Perkin Trans. I (1984) 785, and Tetrahedron Lett. (1990) 31:23

Preparation of Polyhydroxymates Using Chiral .alpha.-Amino Acids AsBuilding Blocks. Polyhydroxymates can be synthesized using techniques asdisclosed in Kolasa, T., and Chimiak, A., Tetrahedron (1977) 33:3285.References cited within Kolasa disclose and describe chemical techniquesfor synthesizing N-hydroxy amino acids which can be used in mimeticsynthesis.

Preparation of β-Polyesters Using Chiral β-Hydroxy Acids as BuildingBlocks. β-polyesters can be synthesized using a synthesis protocol asdescribed in Elliott, J. D., et al., Tetrahedron Lett. (1985) 26:2535,and Tetrahedron Lett. (1974) 15:1333.

Preparation of Polysulfonamides Using Chiral β-Amino Sulfonic Acids asBuilding Blocks. Polysulfonamides can be synthesized using the reactionscheme shown in U.S. Pat. No. 6,075,121. The chiral β-amino acids havebeen described within Kokotos, G., Synthesis (1990) 299.

Preparation of N-Alkylated Polysulfonamides Using Achiral β-AminoSulfonic Acids As Building Blocks. Similarly, these polysulfonamides canbe synthesized using the reaction scheme shown in U.S. Pat. No.6,075,121.

Preparation of Polyureas Using Achiral β-Amino Acids as Building Blocks.Polyureas can be synthesized using techniques such as those described inShiori, T., et al., J. Am. Chem. Soc. (1972) 94:6302, and Scholtz, J.,and Bartlett, P., Synthesis (1989) 542.

Preparation of Polyurethanes Using Achiral β-Amino Alcohols as BuildingBlocks. Polyurethanes can be synthesized using the reaction scheme shownin U.S. Pat. No. 6,075,121. Individual N-substituted glycine analogs areknown in the art, and may be prepared by known methods. See, forexample, Sempuku et al., JP 58/150,562 (Chem Abs (1984) 100:68019b);Richard et al., U.S. Pat. No. 4,684,483; and Pulwer et al., EPO 187,130.

Several N-substituted glycine derivatives are available from commercialsources. For example, N-benzylglycine is available from Aldrich ChemicalCo. (Milwaukee, Wis.) as the ethyl ester. The ester is hydrolyzed inKOH/MeOH, then protonated in HCl to yield N-benzylglycine. This may thenbe protected with Fmoc (fluorenylmethoxycarbonyl) by treatment withFmoc-Cl in aqueous dioxane at high pH (about 10).

Other N-substituted glycine analogs are synthesized by simple chemicalprocedures. N-isobutylglycine may be prepared by reacting excess2-methylpropylamine with a haloacetic acid.

N-(2-aminoethyl)glycine may be prepared by reacting excess1,2-diaminoethane with a haloacetic acid and purifying on Dowex-1® (OHform), eluting with acetic acid. The unprotected amine is protected witht-butoxycarbonyl (t-Boc) using conventional techniques at pH 11.2,followed by protection of the secondary amine with Fmoc.

N-(2-hydroxyethyl)glycine may be prepared by reacting excess2-aminoethanol with haloacetic acid and purifying on Dowex-1® (OH form),eluting with acetic acid. The amine nitrogen is then protected withFmoc. Next, the acid group is esterified with methanol under acidicconditions. The methyl ester is then treated with isobutylene to formthe t-butyl ether. Then, the methyl ester is hydrolyzed using porcineliver esterase in phosphate buffer at pH 8.0, to provide a protectedN-substituted glycine analog in a form suitable for mimetic synthesis.As an alternative to the above, the Fmoc-hydroxyethylglycine is treatedwith t-butyldiphenylsilylchloride in DMF and imidazole to give asilyl-protected alcohol.

N-(carboxymethyl)glycine may be prepared by reacting glycine t-butylester with 2-haloacetate in aqueous solution. The product may beprotected directly by addition of Fmoc. As an alternative, theN-(carboxymethyl)glycine may be prepared by mixing glycine t-butylester, glyoxylic acid and palladium on charcoal under an atmosphere ofhydrogen in water at pH 6. The compound is then treated with FMOC in theusual manner.

Once the monomers have been synthesized, they may be coupled with othermonomers and/or conventional amino acids to form analogs using standardpeptide chemistry. For example, an Fmoc-protected monomer (N-substitutedglycine or conventional amino acid) may be immobilized on a suitableresin (e.g., HMP) by reaction withbenzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate(BOP) or a carbodiimide (for example, dicyclohexylcarbodiimide) underbasic conditions (e.g., pH 9) in a suitable solvent. The Fmoc protectinggroup is removed by treatment with piperidine. Each additional monomeris then attached sequentially using BOP or a carbodiimide, until theentire sequence has been constructed. The completed chain is thendetached from the resin and the sidechain deprotected by treating withtrifluoroacetic acid (TFA).

Alternatively, one may connect N-substituted glycine analogs to the endsof mimetics produced by other methods, for example, by recombinantexpression or isolation from natural sources. Further, N-substitutedglycine analogs may be inserted within the sequence of such mimetics bycleaving the mimetic at the desired position, attaching an N-substitutedglycine analog, and reattaching the remainder of the molecule or achemically-synthesized replacement.

The compositions for administration can take the form of bulk liquidsolutions or suspensions, or bulk powders. More commonly, however, thecompositions are presented in unit dosage forms to facilitate accuratedosing. The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical excipient. Typical unit dosage forms includeprefilled, premeasured ampules or syringes of the liquid compositions orpills, tablets, capsules, losenges or the like in the case of solidcompositions. In such compositions, the mimetic is usually a minorcomponent (from about 0.1 to about 50% by weight or preferably fromabout 1 to about 40% by weight) with the remainder being variousvehicles or carriers and processing aids helpful for forming the desireddosing form.

Suitable excipients or carriers and methods for preparing administrablecompositions are known or apparent to those skilled in the art and aredescribed in more detail in such publications as Remington'sPharmaceutical Science, Mack Publishing Co, NJ (1991). In addition, themimetics may be advantageously used in conjunction with otherchemotherapeutic agents such as diethylstilbestrol or DES,5-fluorouracil, methotrexate, interferon-alpha, asparaginase, tamoxifen,flutamide, etc, and chemotherapeutic agents described in the MerckManuel, 16th edition 1992, Merck Research Laboratories, Rahway, N.J.;Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9^(th)Ed., 1996, McGraw-Hill, esp. Chabner et al., Antineoplastic Agents, etc.or otherwise known in the art. Hence the compositions may beadministered separately, jointly, or combined in a single dosage unit.In a particular embodiment, the combination therapy is effected by aconjugate of the mimetic bound covalently to the anti-neoproliferativechemotherapeutic or other pharmaceutically active agent. Any suitableconjugation chemistry may be used.

The amount administered depends on the mimetic formulation, route ofadministration, etc. and is generally empirically determined in routinetrials, and variations will necessarily occur depending on the target,the host, and the route of administration, etc. Generally, the quantityof active compound in a unit dose of preparation may be varied oradjusted from about 0.1 mg to 1000 mg, preferably from about 1 mg to 300mg, more preferably 10 mg to 200 mg, according to the particularapplication. The actual dosage employed may be varied depending upon therequirements of the patient and the severity of the condition beingtreated. Determination of the proper dosage for a particular situationis within the skill of the art. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small amounts until the optimumeffect under the circumstances is reached. For convenience, the totaldaily dosage may be divided and administered in portions during the dayif desired.

The following are examples (Formulations 1-4) of mimetic capsuleformulations.

TABLE 1 Capsule Formulations Capsule Formulation Formula Formula FormulaFormula 1 2 3 4 mg/ mg/ mg/ mg/ capsule capsule capsule capsule Mimetic(Solid Solution) 100 400 400 200 Silicon Dioxide 0.625 2.5 3.75 1.875Magnesium Stearate NF2 0.125 0.5 0.125 0.625 Croscarmellose Sodium NF11.000 44.0 40.0 20.0 Pluronic F68 NF 6.250 25.0 50.0 25.0 SiliconDioxide NF 0.625 2.5 3.75 1.875 Magnesium Stearate NF 0.125 0.5 1.250.625 Total 118.750 475.00 475.00 475.00 Capsule Size No. 4 No. 0 No. 0No. 2

Preparation of Solid Solution

Crystalline mimetic (80 g/batch) and the povidone (NF K29/32 at 160g/batch) are dissolved in methylene chloride (5000 mL). The solution isdried using a suitable solvent spray dryer and the residue reduced tofine particles by grinding. The powder is then passed through a 30 meshscreen and confirmed to be amorphous by x-ray analysis.

The solid solution, silicon dioxide and magnesium stearate are mixed ina suitable mixer for 10 minutes. The mixture is compacted using asuitable roller compactor and milled using a suitable mill fitted with30 mesh screen. Croscarmellose sodium, Pluronic F68 and silicon dioxideare added to the milled mixture and mixed further for 10 minutes. Apremix is made with magnesium stearate and equal portions of themixture. The premix is added to the remainder of the mixture, mixed for5 minutes and the mixture encapsulated in hard shell gelatin capsuleshells.

The mimetics can be administered by a variety of methods including, butnot limited to, parenteral, topical, oral, or local administration, suchas by aerosol or transdermally, for prophylactic and/or therapeutictreatment. The chemotherapeutic agent and/or radiation therapy can beadministered according to therapeutic protocols well known in the art.It will be apparent to those skilled in the art that the administrationof the chemotherapeutic agent and/or radiation therapy can be varieddepending on the disease being treated and the known effects of thechemotherapeutic agent and/or radiation therapy on that disease. Also,in accordance with the knowledge of the skilled clinician, thetherapeutic protocols (e.g., dosage amounts and times of administration)can be varied in view of the observed effects of the administeredtherapeutic agents (i.e., antineoplastic agent or radiation) on thepatient, and in view of the observed responses of the disease to theadministered therapeutic agents.

The particular choice of mimetic, chemotherapeutic agent and/orradiation depends upon the diagnosis of the attending physicians andtheir judgement of the condition of the patient and the appropriatetreatment protocol. The mimetic, chemotherapeutic agent and/or radiationmay be administered concurrently (e.g., simultaneously, essentiallysimultaneously or within the same treatment protocol) or sequentially,in any order, depending upon the nature of the proliferative disease,the condition of the patient, and the actual choice of chemotherapeuticagent and/or radiation to be administered in conjunction (i.e., within asingle treatment protocol) with the mimetic. Similarly, the mimetic andthe chemotherapeutic agent do not have to be administered in the samepharmaceutical composition, and may, because of different physical andchemical characteristics, be administered by different routes.

In one embodiment of the present invention, the method of the inventionincludes systemic or local administration of a mimetic. Where systemicadministration is desired, the mimetic may be administered, for example,by intravenous injection or orally. One embodiment of the inventionprovides local administration of the mimetic, for example, at the tumorsite. With local administration of the mimetic, the preferred mode ofadministration is by local injection. However, local administration mayalso be by catheter, or by local deposition, for example by intra- orperitumoral administration of products sold under the trademarkDepofoam®, slow release pump/drug delivery service, implantable ortopical gel or polymer, depending on the nature and location of thetumor. Administration of the therapeutics of the invention can also beeffected by gene therapy protocol.

The therapeutics of the invention can be administered in atherapeutically effective dosage and amount, in the process of atherapeutically effective protocol for treatment of the patient. Theinitial and any subsequent dosages administered will depend upon thepatient's age, weight, condition, and the disease, disorder orbiological condition being treated. Depending on the therapeutic, thedosage and protocol for administration will vary, and the dosage willalso depend on the method of administration selected, for example, localor systemic administration. For a very potent mimetic, microgram (ug)amounts per kilogram of patient may be sufficient, for example, in therange of about 1 ug/kg to about 500 mg/kg of patient weight, and about100 ug/kg to about 5 mg/kg, and about 1 ug/kg to about 50 ug/kg, and,for example, about 10 ug/kg.

In general, routine experimentation in clinical trials will determinespecific ranges for optimal therapeutic effect, for each therapeutic,each administrative protocol, and administration to specific patientswill also be adjusted to within effective and safe ranges depending onthe patient condition and responsiveness to initial administrations.However, the ultimate administration protocol will be regulatedaccording to the judgment of the attending clinician considering suchfactors as age, condition and size of the patient as well as mimeticpotency, severity of the disease being treated. For example, a dosageregimen of the mimetics can be oral administration of from 10 mg to 2000mg/day, preferably 10 to 1000 mg/day, more preferably 50 to 600 mg/day,in two to four (preferably two) divided doses, to reduce tumor growth.In cases where the mimetic is based on a fused-ring cyclicbenzocycloheptapyridine, the preferred dosage of the inhibitor is oraladministration of from 50 to 600 mg/day, more preferably 50 to 400mg/day, in two divided doses. Intermittant therapy (e.g., one week outof three weeks or three out of four weeks) may also be used.

In one example of combination therapy in the treatment of pancreaticcancer, the mimetic is administered orally in a range of from 50 to 400mg/day, in two divided doses, on a continuous dosing regimen; and theantineoplastic agent is gemcitabine administered at a dosage of from 750to 1350 mg/m² weekly for three out of four weeks during the course oftreatment. In another example of combination therapy in the treatment oflung cancer, the mimetic is administered orally in a range of from 50 to400 mg/day, in two divided doses, on a continuous dosing regimen; andthe antineoplastic agent is paclitaxel administered at a dosage of from65 to 175 mg/m² once every three weeks. In another example ofcombination therapy in the treatment of gliomas, the mimetic isadministered orally in a range of from 50 to 400 mg/day, in two divideddoses; and the antineoplastic agent is temozolomide administered at adosage of from 100 to 250 mg/m². In another example of combinationtherapy, the mimetic is administered orally in a range of from 50 to 400mg/day, in two divided doses, on a continuous dosing regimen; and theantineoplastic agent is 5-Fluorouracil (5-FU) administered either at adosage of 500 mg/m² per week (once a week), or at a dosage of 200-300mg/m² per day in the case of continuous infusion of the 5-FU. In thecase of 5-FU administration on a weekly injection, 5-FU may beadministered in combination with a foliate agonist, e.g., Leucovoran (ata dosage of 20 mg/m²/week).

A preferred embodiment of the invention includes monitoring the effectsof the treatment with a mimetic for signs of tumor regression, andsubsequently adjusting the administration of further doses accordingly.For example, a person with breast carcinoma would be treated locallywith an agent such as cyclophosphamide methotrexate 5-FU (CMF) ortamoxifen or local radiation therapy and a mimetic. Subsequentmammography, ultrasound, or physical exams, as compared with the samepre-treatment tests, would direct the course and dosage of furthertreatment.

The attending clinician, in judging whether treatment is effective atthe dosage administered, will consider the general well-being of thepatient as well as more definite signs such as relief of disease-relatedsymptoms, inhibition of tumor growth, actual shrinkage of the tumor, orinhibition of metastasis. Size of the tumor can be measured by standardmethods such as radiological studies, and successive measurements can beused to judge whether or not growth of the tumor has been retarded oreven reversed. Relief of disease-related symptoms such as pain, andimprovement in overall condition can also be used to help judgeeffectiveness of treatment. Accordingly, preferred embodiments of theinvention include monitoring of the patient after treatment with amimetic for signs of tumor regression. Such monitoring includes but isnot limited to physical exam, CT scan, MRI, mammography, chest X-rays,bone scans, ultra-sounds, bronchoscopy, endoscopy, colonoscopy,laparoscopy, and tests for tumor markers such as PSA, CEA, and CA125.The appropriateness of any form of monitoring will be determined by thenature of the cancer being treated.

EXAMPLES

The following examples are used to assay for bioactivity of Smacmimetics, e.g. as measured by IAP binding, procaspase-3 activation orpromotion of apoptosis. These assays may also be used to screen foragents (e.g. antagonists) which potentiate such mimetic activity.

Example 1 In Vitro IAP (BIR) Binding/Interaction Assay

Interaction between mimetics and IAPs was examined by GST-mediatedpull-down assays. Approximately 0.4 mg of a recombinant IAP fragment(second and third BIR motifs of XIAP) is bound to 200 ml of glutathioneresin as a GST-fusion protein and incubated with 0.5 mg of radiolabeledmimetics at room temperature. After extensive washing with an assaybuffer containing 25 mM Tris, pH 8.0, 150 mM NaCl, and 2 mMdithiothreitol (DTT), the complex is eluted with 5 mM reducedglutathione and visualized by SDS-PAGE with Coomassie staining. Thisassay demonstrates that the tested mimetics specifically bind IAP.

Example 2 High-Throughput In Vitro Fluorescence Polarization BindingAssay Sensor: Rhodamine-labeled mimetic (final conc.=1-5 nM) Receptor:Glutathione-S-transferase/BIR2,3 fusion protein (final conc.=100-200 nM)Buffer: 10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH 7.6

-   1. Add 90 microliters of mimetic/BIR2,3 mixture to each well of a    96-well microtiter plate.-   2. Add 10 microliters of test compound per well.-   3. Shake 5 min and within 5 minutes determine amount of fluorescence    polarization by using a Fluorolite FPM-2 Fluorescence Polarization    Microtiter System (Dynatech Laboratories, Inc).

Tested mimetics significantly and specifically bind the IAP BIR2,3domain.

-   3. High throughput solid phase mimetic-BIR2,3    binding/binding-interference assay.

A. Reagents:

-   -   Neutralite Avidin: 20 μg/ml in PBS.    -   Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at room        temperature.    -   Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl₂, 1%        glycerol, 0.5% NP-40, 50 mM b-mercaptoethanol, 1 mg/ml BSA,        cocktail of protease inhibitors.    -   ³³P mimetic 10× stock: 10⁻⁸-10⁻⁶ M “cold” mimetic supplemented        with 200,000-250,000 cpm of labeled mimetic (Beckman counter).        Place in the 4° C. microfridge during screening.    -   Protease inhibitor cocktail (1000×): 10 mg Trypsin Inhibitor        (BMB #109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine        (Sigma #B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF        (BMB #917575), and 2 mM NaVO₃ (Sigma #S-6508) in 10 ml of PBS.    -   BIR2,3: 10⁻⁷-10⁻⁵ M biotinylated BIR2,3 domain (supra) in PBS.        B. Preparation of assay plates:    -   Coat with 120 μl of stock N-Avidin per well overnight at 4° C.    -   Wash 2 times with 200 μl PBS.    -   Block with 150 μl of blocking buffer.    -   Wash 2 times with 200 μl PBS.

C. Assay:

-   -   Add 40 μl assay buffer/well.    -   Add 10 μl compound or extract.    -   Add 10 μl ³³P-mimetic (20-25,000 cpm/0.1-10        pmoles/well=10⁻⁹-10⁻⁷ M final conc)    -   Shake at 25° C. for 15 minutes.    -   Incubate additional 45 minutes at 25° C.    -   Add 40 μM biotinylated BIR2,3 (0.1-10 pmoles/40 ul in assay        buffer)    -   Incubate 1 hour at room temperature.    -   Stop the reaction by washing 4 times with 200 μM PBS.    -   Add 150 μM scintillation cocktail.    -   Count in Topcount.

D. Controls for all assays (located on each plate):

-   -   a. Non-specific binding    -   b. Soluble (non-biotinylated BIR2,3) at 80% inhibition

Mimetics significantly and specifically bind the IAP BIR2,3 domain.Example 4 Hela Cell Extracts: Radiolabeled Procaspase-3 Activation Assay

20 mg S-100 extracts of HeLa cells were incubated alone (Control), orwith mimetics (50 nM) nM, or with 30-1000 mM of N-terminal Smac peptidesin different lengths. The reactions were carried out with the additionof 1 mM dATP, 1 mM additional MgCl2, 0.2 mg/ml horse heart cytochrome c,and 1 ml of in vitro translated, ³⁵S-labeled caspase-3 in a final volumeof 20 ml. The reaction mixtures were incubated at 30° C. for 1 hrfollowed by electrophoresis on a 15% PAGE gel. The gel was subsequentlytransferred onto a nitrocellulose filter and exposed to a phosphoimagingcassette. Mimetics and Smac fragments significantly promoted activationof procaspase-3, whereas negative control Smac-7R did not.

Example 5 Hela Cell Extracts: Spectrofluorometric Procaspase-3Activation Assay

Human Hela S3 cells were cultured in 150-mm tissue culture dishes inDMEM medium (Dulbecco's modified eagle's medium containing 100 U/ml ofpenicillin and 100 ug/ml of streptomycin sulfate) supplemented with 10%(v/v) fetal calf serum, and grown in monolayer at 37° C. in anatmosphere of 5% CO₂. Cells at 70% confluence were washed once with 1×phosphate-buffered saline (PBS) and harvested by centrifugation at 800×gfor 5 min at 4° C. The cell pellets were resuspended in 3 volume ofBuffer A (20 mM Hepes-KOH, pH 7.5, 10 mM KCL, 1.5 mM MgCl2, 1 mM sodiumEDTA, 1 mM sodium EGTA, 1 mM DTT, and 0.1 mM PMSF). Human c-IAP-1 orc-IAP-2, or XIAP either full length of truncated proteins that containthe first three BIR domains, or the second and third BIR domains areadded to the HeLa cell extracts and the caspase activation reaction isstarted by adding 1 mM dATP and 300 nM cytochrome c. The caspase-3activity is measured by spectrofluorometric assay as previouslydescribed by MacFarlane et al. (1997, J. Cell Biol. 137, 469-479).Aliquots of 8 mg of S-100 prepared as in Liu et al. were assayed in96-well microtiter format in a 150 ml of reaction containing 0.1 mMHepes, PH 7.4, 2 mM DTT, 0.1% (w/v) Chaps, and 1% (w/v) Sucrose. Thereactions were started by adding caspase specific fluorogenic substrate(Enzyme Systems, CA) to the final concentration of 20 mM and continuedat 37° C. for 30 min. Liberation of AFC from the substrates wasmonitored continuously using excitation/emission wavelength pairs of400/505 nm. Mimetics and Smac fragments (except 7R) significantlypromoted activation of procaspase-3.

Example 6 Reconstituted Recombinant Radiolabeled Procaspase-3 ActivationAssay

Mimetics and N-terminal Smac peptides (30-3000 mM) were incubated withrecombinant human Apaf-1 (40 nM), recombinant human procaspase-9 (2 nM),purified horse heart cytochrome c (nM) and mouse XIAP (70 nM) in thepresence of 1 mM dATP, 1 mM MgCl₂ and 1 ml of in vitro translated,³⁵S-labeled caspase-3 in a final volume of 20 ml. The reaction mixtureswere incubated at 30° C. for 1 hr followed by electrophoresis on a 15%PAGE gel. The gel was transferred onto a nitrocellulose filter andexposed to a phosphoimaging cassette. Active Smac protein (50 nM) and aninactive peptide Smac-7R (3000 mM) are also included as controls.Mimetics and Smac fragments (except 7R) significantly promotedactivation of procaspase-3.

Example 7 Reconstituted Recombinant Spectrofluorometric Procaspase-3Activation Assay

A reconstituted recombinant procaspase-3 activation system isconstructed as described above except the human caspase-3 is producedfrom bacterial expression as described in Liu et al., 1997 (supra) andis not labeled. The caspase-3 activity is measured byspectrofluorometric assay as previously described by MacFarlane et al.(1997, J. Cell Biol. 137, 469-479). Aliquots of 8 mg of S-100 preparedas above were assayed in 96-well microtiter format in a 150 ml ofreaction containing 0.1 mM Hepes, PH 7.4, 2 mM DTT, 0.1% (w/v) Chaps,and 1% (w/v) Sucrose. The reactions were started by adding a caspasespecific fluorogenic substrate (Enzyme Systems, CA) to the finalconcentration of 20 mM and continued at 37° C. for 30 min. Liberation ofAFC from the substrates was monitored continuously usingexcitation/emission wavelength pairs of 400/505 nm. Mimetics and Smacfragments (except 7R) significantly promoted activation of procaspase-3.

Example 8 Cell-Based Assay: Smac Peptides Potentiate Apoptosis Inducedby UV or Etoposide in Cultured HeLa Cells

0.75×105 of HeLa—S cells/well were plated in 48-well tissue cultureplate. Cells were incubated with 1 mM inactive Smac peptide or with 1 mMN-terminal 4-amino acid Smac peptide, with selected mimetics, or withvehicle only (Control) for 12 hr. The cells were then treated witheither 320,000 microjoules of UV irradiation using a Stratalinker orwith 100 mM chemotherapeutic Etoposide. Cells were then stained with 1mg/ml Hoechst 33342 dye at different time points and apoptotic cellswere counted as those with condensed nuclear chromatin under afluorescent microscopy. Mimetics, including wild-type Smac peptides,showed significant increases in apoptotic induction at 2, 4 and 6 hrs(for UV insult) and at 10 and 20 hr (for etoposide).

Example 9 In Vivo Metastasis Assay

Immunosuppressed mice (athymic nude/nude SCID females from HarlanSprague Dawley) are housed in autoclaved cages with microisolator tops,and all manipulations of the animals are done in a laminar flow hoodafter wiping down both the hood, gloves and cages with ABQ sterilant.The mice are fed sterile Pico Lab Chow (Purina) and autoclaved St. Louistap water. Mimetics are administered intra-gastrically daily to the micein sterile water containing 2% carboxymethyl cellulose via sterile,disposable animal feeding needles (Poper & Sons Cat #9921; 20 g×1.5″),seven days a week between 7:00 and 8:00 am. The compounds and control(sterile water plus 2% carboxymethyl cellulose) are kept stored at −80°C. wrapped in aluminum foil to prevent any light induced changes, andeach day's supply is thawed just prior to use.

Compounds are tested for their effects on the metastatic potential ofC8161 cells injected intravenously via the tail vein: at 40 and 100mg/kg, compared to the control. The concentration of the compounds inthe vials used to give the 100 mg/kg doses are 2.5 times that in the 40mg/kg dose so that approximately the same volume is used in both cases,approximately 0.5 mL/animal. The experiments start with nine animals pergroup at day −4. On day zero, 2×10⁵ C8161 cells in cold Hank's BalancedSalt Solution (HBSS) are injected intravenously via tail veininoculation. The protocol is continued for an additional 24 days, atwhich time the animals are sacrificed and their lungs removed and fixedin a solution of Bouins/formaldehyde (5 parts: 1 part). Tumors arequantified on the entire surface of the lungs by rotating the lungs andcounting the tumors on each lobe using a 6× magnifying glass.Statistical analysis is performed using the statistical package ofMicrosoft's Excel spreadsheet software.

The effects of test mimetics, at two different concentrations, on themetastatic potential of C8161 cells in SCID mice are evaluated: oralgavaging of the animals with mimetics significantly reduces the numberof lung metastases in the SCID mouse population.

Example 10 In Vivo Combination Therapy: B.I.D. & Q.I.D.

The effect of in vivo combination therapy of mimetics (20 or 80mpk/dosing p.o. or i.p.) with chemotherapies paclitaxel (5 or 20 mpk),5-Fu (50 mpk), vincristine (1 mpk) or cytoxan (100 mpk, BID, ip) on HTB177 xenografts (NCI-H460, a human lung large cell carcinoma) using twoand four times a day dosing is demonstrated in athymic nu/nu femalemice, 5-6 weeks old. On Day 0, HTB 177 cells, 3×10⁶, are injected s.c.into the flank of 220 mice and the mice divided into treatment andcontrol groups:

Mimetics were dissolved in 20% hydroxyl-propyl-betacyclodexatrin(Vehicle I); 0.2 ml of mimetic solution was the dosing volume.Paclitaxel was dissolved in a diluted ethanol/cremophor EL solution(Vehicle II) and the i.p. dosing volume for paclitaxel was 0.1 ml.Cytoxan, 5-FU, and Vincristine were dissolved in sterile water. The 80mpk dosing mimetic solution is made by adding 17 ml of 20% HPBCD to a 50ml tube containing 136 mg of mimetic to dissolve. The mixture wassonicated until a complete solution was made. The 20 mpk dosing solutionis made by placing 2 ml of the 80 mpk solution into a 15 ml tube, adding6 ml of 20% HPBCD, and vortexing the solution to mix it.

Tumor cells are inoculated into mice in the morning of Day 0, and themice weighed, randomized, and ear-marked afterwards. Drug treatmentbegins at 7:30 am on Day 4. The animals are dosed with mimetic orvehicle I solution, at 7:30 am, and 7:30 pm, 7 days a week. Tumor growthis quantitated by measuring tumor volume on Day 7 and Day 14. Bothmimetic and chemotherapies demonstrate inhibition; combination therapiesprovide enhanced inhibition over either therapy alone.

Example 11 In Vivo Therapy in the WAP-RAS Transgenic Model

Mimetic and Paclitaxel combination efficacy is also evaluated in theWap-ras transgenic model. This model is used in a therapeutic mode inwhich treatments are initiated after mice had well developed tumors.

Mimetics (20 mpk/dosing po) were dissolved in 20%hydroxyl-propyl-betacyclodexatrin (Vehicle I). 0.2 ml of mimeticsolution is the oral dosing volume. Paclitaxel (5 mpk/dosing ip) wasdissolved in a diluted ethanol/cremophor EL solution (vehicle II) andthe i.p. dosing volume for paclitaxel was 0.1 ml.

The mice are weighed, randomized, and ear-marked on Day 0. Mimetictreatment and Vehicle I treatment began on Day 1 and continued every 12hours until Day 21. Paclitaxel and Vehicle II treatments are started onDay 4 and continued daily on Day 5, 6, and 7. Wap-ras tumors do notrespond to treatment with Paclitaxel but do respond to mimetic treatmentat 20 mpk alone and combined therapy enhanced efficacy.

All publications and patent applications cited in this specification andall references cited therein are herein incorporated by reference as ifeach individual publication or patent application or reference werespecifically and individually indicated to be incorporated by reference.Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

Exemplary Dimers

-   -   R1 and R1′ can be the same or different and include H; Me    -   R2 and R2′ can be the same or different and include Me; Et    -   R7 and R7′ can be the same or different and include aryl,        heteroaryl, CH2aryl, CH2heteroaryl, branched alkyl, cycloalkyl,        functionalized alkyl or cycloalkyl (functionalization may        include, inter alia, unsaturation, heteroatoms, aryl,        heteroaryl)    -   Q and Q′ can be the same or different and include O, S, NR1

-   -   R1 and R1′ can be the same or different and include H; Me    -   R2 and R2′ can be the same or different and include Me; Et    -   R7 and R7′ can be the same or different and include aryl,        heteroaryl, CH2aryl, CH2heteroaryl, branched alkyl, cycloalkyl,        functionalized alkyl or cycloalkyl (functionalization may        include, inter alia, unsaturation, heteroatoms, aryl,        heteroaryl)    -   Q and Q′ can be the same or different and include O, S, NR1

-   -   R^(4′) can include:

-   -   R and R′ can be the same or different and include CH2; CHMe;        CHEt; CMe2; 1,1-disubstituted cyclopropyl    -   R1 and R1′ can be the same or different and include H; Me    -   R2 and R2′ can be the same or different and include Me; Et    -   R3 and R3′ can be the same or different and include H; Me;        halogen; straight chain or branched alkyl; cycloalkyl; aryl;        heteroaryl; functionalized alkyl or cycloalkyl        (functionalization may include, inter alia, unsaturation,        heteroatoms, aryl and heteroaryl); OR5; NR5R6    -   R4 can be chosen from R5 and R4′    -   R5 and R6 can include H, Me, straight chain alkyl, or any of R7    -   R7=aryl, heteroaryl, branched alkyl, cycloalkyl, functionalized        alkyl or cycloalkyl (functionalization may include, inter alia,        unsaturation, heteroatoms, aryl, heteroaryl)    -   R8=CH2OR5, CH2OC(O)R5, CH2OC(O)NHR5, CO2R5, C(O)NR5R6    -   R9=H, OR5, NR5R6    -   A, B, and Z includes CH, N, C—R7    -   Q=CH2, CHMe, O, S, NR5    -   X and X′ can be the same or different and include NH, O, CH2    -   Y═CH2, C(O), C(S), CHMe    -   LINKER is a contiguous chain that can incorporate substitution,        heteroatoms, unsaturation (alkene, alkyne), cyclic, aromatic and        heteroaromatic fragments.

Examples include, but are not limited to:

Examples of Unsymmetrical Linkers:

Examples of Truncated Linkers

-   -   R^(4′) can include:

-   -   R and R′ can be the same or different and include CH2; CHMe;        CHEt; CMe2; 1,1-disubstituted cyclopropyl    -   R1 and R1′ can be the same or different and include H; Me    -   R2 and R2′ can be the same or different and include Me; Et    -   R3 and R3′ can be the same or different and include H; Me;        straight chain or branched alkyl; cycloalkyl; functionalized        alkyl or cycloalkyl (functionalization may include, inter alia,        unsaturation, heteroatoms, aryl and heteroaryl)    -   R4 can be chosen from R5 and R4′    -   R5 and R6 can include H, Me, straight chain alkyl, or any of R7    -   R7=aryl, heteroaryl, branched alkyl, cycloalkyl, functionalized        alkyl or cycloalkyl (functionalization may include, inter alia,        unsaturation, heteroatoms, aryl, heteroaryl)    -   R8=CH2OR5, CH2OC(O)R5, CH2OC(O)NHR5, CO2R5, C(O)NR5R6    -   R9=H, OR5, NR5R6    -   A, B, and Z includes CH, N, C—R7    -   Q=CH2, CHMe, O, S, NR5    -   X and X′ can be the same or different and include NH, O, CH2    -   Y═CH2, C(O), C(S), CHMe    -   LINKER is a contiguous chain that can incorporate substitution,        heteroatoms, unsaturation (alkene, alkyne), cyclic, aromatic and        heteroaromatic fragments.

Examples include, but are not limited to:

-   -   R^(4′) can include:

-   -   R1 and R1′ can be the same or different and include H; Me    -   R2 and R2′ can be the same or different and include Me; Et    -   R4 can be chosen from R5 and R4′    -   R5 and R6 can include H, Me, straight chain alkyl, or any of R7    -   R7=aryl, heteroaryl, branched alkyl, cycloalkyl, functionalized        alkyl or cycloalkyl (functionalization may include, inter alia,        unsaturation, heteroatoms, aryl, heteroaryl)    -   R8=CH2OR5, CH2OC(O)R5, CH2OC(O)NHR5, CO2R5, C(O)NR5R6    -   R9=H, OR5, NR5R6    -   A, B, and Z includes CH, N, C—R7    -   Q and Q′ can be the same or different and include CH2, CHMe, O,        S, NR5    -   X and X′ can be the same or different and include NH, O, CH2    -   Y═CH2, C(O), C(S), CHMe    -   LINKER is a contiguous chain that can incorporate substitution,        heteroatoms, unsaturation (alkene, alkyne), cyclic, aromatic and        heteroaromatic fragments.

Examples include, but are not limited to:

-   -   R^(4′) can include:

-   -   R and R′ can be the same or different and include CH2; CHMe;        CHEt; CMe2; 1,1-disubstituted cyclopropyl    -   R1 and R1′ can be the same or different and include H; Me    -   R2 and R2′ can be the same or different and include Me; Et    -   R3 and R3′ can be the same or different and include H; Me;        straight chain or branched alkyl; cycloalkyl; functionalized        alkyl or cycloalkyl (functionalization may include, inter alia,        unsaturation, heteroatoms, aryl and heteroaryl)    -   R4 can be chosen from R5 and R4′    -   R5 and R6 can include H, Me, straight chain alkyl, or any of R7    -   R7=aryl, heteroaryl, branched alkyl, cycloalkyl, functionalized        alkyl or cycloalkyl (functionalization may include, inter alia,        unsaturation, heteroatoms, aryl, heteroaryl)    -   R8=CH2OR5, CH2OC(O)R5, CH2OC(O)NHR5, CO2R5, C(O)NR5R6    -   R9=H, OR5, NR5R6    -   A, B, and Z includes CH, N, C—R7    -   Q=CH2, CHMe, O, S, NR5    -   X and X′ can be the same or different and include NH, O, CH2    -   Y═CH2, C(O), C(S), CHMe    -   LINKER is a contiguous chain that can incorporate substitution,        heteroatoms, unsaturation (alkene, alkyne), cyclic, aromatic and        heteroaromatic fragments.

Examples include, but are not limited to:

-   -   R^(4′) can include:

-   -   R and R′ can be the same or different and include CH2; C(O)    -   R1 and R1′ can be the same or different and include H; Me    -   R2 and R2′ can be the same or different and include Me; Et    -   R4 can be chosen from R5 and R4′    -   R5, R5′, R6, R6′ can be the same or different and include H, Me,        straight chain alkyl, or any of R7    -   R7=aryl, heteroaryl, branched alkyl, cycloalkyl, functionalized        alkyl or cycloalkyl (functionalization may include, inter alia,        unsaturation, heteroatoms, aryl, heteroaryl)    -   R8=CH2OR5, CH2OC(O)R5, CH2OC(O)NHR5, CO2R5, C(O)NR5R6    -   R9=H, OR5, NR5R6    -   A, B, and Z includes CH, N, C—R7    -   Q=CH2, CHMe, O, S, NR5    -   X and X′ can be the same or different and include NH, O, CH2    -   Y═CH2, C(O), C(S), CHMe    -   LINKER is a contiguous chain that can incorporate substitution,        heteroatoms, unsaturation (alkene, alkyne), cyclic, aromatic and        heteroaromatic fragments.

-   -   R═H, alkyl, branched alkyl

Exemplary Monomers

Experimental Procedure

(S)-Boc-2-[5-(Toluene-r-sulfonyl)-tetrazol-1-ylmethyl]-pyrrolidine) (1)

A sealed tube was charged with p-toluenesulfonyl cyanide (5.0 g, 22.1mmol) and (S)-Boc-2-azido methyl pyrrolidine³ (4.0 g, 22.1 mmol). Themixture was stirred at 80° C. for 40 hrs. The crude product was purifiedon flash silica gel column with (Hexane:EtOAc=1:1) to furnish 1 (7.6 g,84%) as colorless foam. [α]_(D) −11.7 (c 0.64, CHCl₃). ¹H NMR (400 MHz,CD₃OD, −20° C., rotamers 6:4): δ 1.20 & 1.35 (s, 9H), 1.83 & 2.01 (m,4H), 2.50 (s, 3H) 3.40 & 3.45 (m, 2H), 4.40 & 4.43 (m, 1H), 4.71 & 4.82(m, 2H), 7.50 & 7.70 (d, J=8 Hz, 2H), 8.00 & 8.40 (d, J=8 Hz, 2H); ¹³CNMR (75 MHz, CDCl₃): δ 22.0, 23.7, 28.4, 46.5, 52.1, 56.1, 76.9, 80.6,129.4, 134.4, 147.4, 154.4, 154.8, 155.5; IR (film): 2976, 1693, 1392,815, 704 cm⁻¹; MS: (ESI) [M+1]⁺ 408.2.

(S)-Boc-2-(5-Phenylsulfanyl-tetrazol-1′-ylmethyl)-pyrrolidine (2)

The mixture of 1 (6.0 g, 14.7 mmol), phenylthiol (6.5 g, 58.8 mmol) andK₂CO₃ (4.5 g, 41.1 mmol) in CH₃CN (74 ml) was stirred for 2 days at roomtemperature. The reaction mixture was filtered through a pad of Celiteand then concentrated in vacuo. The crude was purified by silica gelcolumn with (Hexane:EtOAc=1:1) as an eluant to give 2 (5.2 g, 97%) as ancolorless oil. [α]_(D) −29.2 (c 1.34, CHCl₃). ¹H NMR (400 MHz, CD₃OD,−20° C., rotamers 1:1): δ 1.30 & 1.42 (s, 9H), 1.73 & 2.01 (m, 4H) 3.40& 3.45 (m, 13.2 Hz, 2H), 4.23 & 4.25 (m, 1H), 4.38 & 4.58 (m, 2H), 7.42& 7.46 (m, 3H) 7.56 & 7.60 (m, 2H); ¹³C NMR (CDCl₃, 75 MHz): δ 21.2,23.6, 28.5, 46.5, 49.7, 50.4, 56.7, 60.5, 80.7, 128.1, 129.9, 133.1,153.5, 154.9; IR (film): 2975, 1693, 1391, 1167, 688 cm⁻¹; MS: (ESI)[M+1]⁺ 361.2

(S)-2-(5-phenylsulfanyl-tetrazol-1-ylmethyl)-pyrrolidine (3)

2 (5.0 g, 13.8 mmol) was treated with 50% TFA in CH₂Cl₂ (69 ml) for 15min at room temperature. The mixture was sequestered with sat.NaHCO₃ andextracted with CH₂Cl₂ (3×20 ml). The combined extraction were dried overNa₂SO₄, filtered and concentrated. The crude was purified over flashsilica gel column with (CH₂Cl₂:MeOH:NH₄OH=20:0:9:1) as an eluant tofurnish 3 (2.5 g, 69%). [α]_(D) +9.9 (c 1.14, CHCl₃). ¹H NMR (400 MHz,CDCl₃): δ 1.45 (m, J=2 Hz, 8.4 Hz, 1H), 1.80 (m, 2H), 1.95 (m, 1H), 2.5(bs, 1H), 2.99 (t, J=1.6 Hz, 2H), 3.60 (m, 1H), 4.20 (dd, J=8.0 Hz, 13.0Hz, 1H), 4.30 (dd, J=8.0 Hz, 13.0 Hz 1H), 7.40 (m, 3H), 7.58 (m, 2H);¹³C NMR (75 MHz, CDCl₃): δ 23.5, 28.6, 46.1, 47.8, 58.2, 127.2, 130.0,130.2, 133.1, 153.4; IR (film): 3350, 2961, 2871, 1442, 1389, 746, 688cm⁻¹; MS: (ESI) [M+1]⁺ 262.2.

{1-[2-(5-Phenylsulfanyl-tetrazol-1-ylmethyl)-pyrrolidine-1-carbonyl]-2-ynyloxy-propyl}-carbamicacid allyl ester (4)

To a cold solution of 3 (2.5 g, 9.5 mmol) and N-Alloc-(propargyl)Threonine-(2S,3R) (2.7 g, 11.4 mmol) in DMF (48 ml) were added DIPEA(3.3 ml, 19.0 mmol) and HATU (5.4 g, 14.2 mmol). After stirring for 30min at 0° C., the reaction mixture was diluted with Et₂O. To the mixturewas added sat.NaHCO₃ and then separated. The aqueous phase was extractedwith Et₂O (3×30 ml). The combined extraction were washed with 5% HCl (25ml), H₂O (25 ml), sat.NaHCO₃ (20 ml), and Brine (30 ml), and finallydried over MgSO₄, filtered and concentrated in vacuo. The crude waspurified by silica gel column with (hexane:EtOAc=1:1) to afford 4 (3.2g, 70%) as an oil. [α]_(D) −12.2 (c 1.30, CHCl₃) ¹H NMR (400 MHz,CDCl₃): δ 1.22 (d, J=6 Hz, 3H), 1.75-1.90 (m, 4H), 2.50 (t, J=2.4 Hz,1H), 3.70 (m, 2H) 4.00 (m, 1H), 4.20 (dq, J=5.6 Hz, 18.4 Hz, 2H), 4.31(m, 2H), 4.50 (m, 1H), 4.55 (m, 2H), 4.62 (dd, J=4.2 Hz, 1H), 5.20 (d,J=9.6 Hz, 1H), 5.23 (d, J=10.2 Hz, 1H), 5.50 (d, J=8 Hz, 1H), 5.90 (m,1H), 7.40 (m, 3H), 7.60 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 16.0, 24.3,27.5, 47.9, 48.5, 56.5, 56.7, 66.1, 74.2, 74.9, 75.0, 79.8, 117.9,127.6, 129.9, 130.0, 132.7, 133.3, 153.5, 156.4, 169.8; IR (film): 3292,2979, 1715, 1644, 1513, 1442, 1073, 750, 688 cm⁻¹; MS: (ESI) [M+1]⁺485.2.

{1-[2-(5-Phenylsulfanyl-tetrazol-1-ylmethyl)-pyrrolidine-1-carbonyl]-2-ynyloxy-propyl}-ethyl)-carbamicacid 9H-fluoren-9-ylmethyl ester (5)

To a solution of Fmoc-N(Me) L-Ala (6 g, 18.5 mmol) and HOBt (2.5 g, 18.5mmol) in CH₂Cl₂ (60 ml) was added EDCI (3.5 g, 18.5 mmol) at 0° C.stirred for 1 hr and an additional hour at room temperature. To thereaction mixture was added Pd (PPh₃)₄ (3.6 g, 3.0 mmol), a solution of 4(3 g, 6.2 mmol) in CH₂Cl₂ (30 ml) and DABCO (3.7 g, 31 mmol) at roomtemperature followed by stirred for 15 min. The reaction mixture wasconcentrated in vacuo and purified by silica gel column with(hexane:EtOAc=1:1) to furnish 5 (3.8 g, 89%) as pale yellow foam.[α]_(D) −31.9 (c 1.56, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 1.20 (d, J=3.6Hz, 3H), 1.41 (d, J=4.2 Hz, 3H), 1.75-1.98 (bm, 4H), 2.50 (bs, 1H), 2.95(s, 3H), 3.60 (bm, 1H), 3.70 (bm, 1H), 4.01 (bm, 1H), 4.10 (dq J=5.6 Hz,18.1 Hz, 2H), 4.20 (bm, 1H), 4.25 (bm, 1H), 4.28 (bm, 2H) 4.32 (bm, 1H),4.40 (bm, 2H), 4.59 (dd, J=4.2, 8.0 Hz, 1H), 6.81 (bd, 1H), 7.28 (bm,2H), 7.34 (bm, 5H), 7.52 (br m, 4H), 7.72 (d, J=7.6 Hz, m, 2H); ¹³C NMR(75 MHz, CDCl₃): δ 16.1, 24.3, 27.5, 47.4, 48.5, 55.3, 56.4, 68.2, 74.0,75.0, 79.8, 120.1, 125.2, 125.3, 127.2, 127.6, 127.9, 128.6, 129.9,130.1, 132.2, 132.4, 133.3, 141.5, 153.5, 169.3, 171.4; IR (film): 3297,2978, 1693, 1650, 1442, 1400, 1312, 1157, 10948, 758, 742 cm⁻¹; MS:(ESI) [M+1]⁺ 708.2

{1-[2-Bis-(5-Phenylsulfanyl-tetrazol-1-ylmethyl)-pyrrolidine-1-carbonyl]-2-ynyloxy-propyl}-ethyl)-carbamicacid 9H-fluoren-9-ylmethyl ester (6)

A mixture of 5 (3.5 g, 4.9 mmol) and Cu(OAc)₂ (6.2 g, 34.3 mmol) inCH₃CN was refluxed for 30 min. The organic solvent was stripped off andthe Cu(II) salts were removed by filtering over a short pad of silicagel eluting with (CH₂Cl₂/MeOH 9:1) to give a crude, which was completelydried off to furnish crude 6 (3.3 g, 94%) as pale yellow foam. A smallportion of the crude was purified for spectral analysis. [α]_(D) −13.8(c 1.41, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 1.14 (d, J=6 Hz, 6H), 1.34(d, J=7.2 Hz, 6H), 1.74-1.98 (bm, 8H), 2.84 (s, 6H), 3.60 (bm, 4H), 3.87(bm, 2H), 4.09 (ABq, J=16.8 Hz, 4H), 4.20 (bm, 2H), 4.24 (m, 2H), 4.31(bm, 4H), 4.36 (m, 4H), 4.60 (dd, J=4.2 Hz, 2H), 4.69 (dd, J=4, 8.8 Hz,2H), 6.80 (bm, 2H), 7.28 (bm, 4H), 7.34 (bm, 10H), 7.52 (bm, 8H), 7.72(d, J=7.6 Hz, m, 4H); ¹³C NMR (75 MHz, CDCl₃): δ 16.3, 24.3, 27.5, 47.4,48.5, 55.3, 56.4, 68.2, 74.0, 75.0, 79.8, 120.1, 125.2, 125.3, 127.2,127.6, 127.9, 128.6, 129.9, 130.1, 132.2, 132.4, 133.3, 141.5, 153.5,169.3, 171.4; IR (film): 2920, 2850, 1687, 1643, 1441, 1311, 1155, 1083,741 cm⁻¹; MS: (ESI) [M+1]⁺ 1412.2

Bis-2-Methylamino-N-(1-{1-[2-(5-phenylsulfanyl-tetrazol-1-ylmethyl)-pyrrolidine-1-carbonyl]-2-ynyloxy-propyl}-propionamide(7)

A solution of 6 (3 g, 2.1 mmol) was treated with 20% CH₂Cl₂— piperidine(21 ml) for 5 min. The mixture was concentrated in vacuo and thenpurified by silica gel column with (CH₂Cl₂: MeOH: NH₄OH=8:2:0.5) tofurnish 7 (1.4 g 70%) as gummy foam. [α]_(D) −7.4 (c 0.23, CHCl₃). ¹HNMR (400 MHz, CDCl₃): δ 1.19 (d, J=6.4 Hz, 6H), 1.28 (d, J=7.2 Hz 6H),1.78 (m, 4H), 1.90 (m, 4H), 2.50 (s, 6H), 3.05 (q, J=7.2 Hz, 2H), 3.60(bm, 2H), 3.70 (m, 2H), 4.01 (m, 2H), 4.20 (ABq, J=18.4 Hz, 4H), 4.43(dd, J=4.0 Hz & 13.6 Hz, 2H), 4.50 (m, 2H), 4.70 (dd, J=4 Hz & 13.6 Hz,2H), 4.75 (dd, J=4, 8.8 Hz, 2H), 7.40 (m, 6H), 7.60 (m, 4H), 7.82 (d,J=8.4 Hz, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 16.2, 19.6, 22.8, 24.4, 27.5,31.8, 35.2, 47.8, 48.5, 54.6, 56.3, 56.9, 60.4, 70.4, 74.4, 76.1, 76.8,127.7, 129.9, 130.0, 133.3, 153.6, 169.5, 175.4; IR (film): 3339, 2975,1644, 1513, 1428, 1086, 751, 667 cm⁻¹; MS: (ESI) [M+1]⁺ 969.4

REFERENCES

-   1. Lumma, W. C.; Wohl, Ronald A. Eur. Pat. Appl. 1985, EP 134424 A1-   2. Bejjani, J; Chemla, F; Audouin, M. J. Org. Chem., 2003, 68,    9747-9752.-   3. Black, Julian; Brown, Alan D; Erizabet C L; Smith, J D; Mckelloy,    A B. 2000, JP 2000063380-   4. Dininno, F; Guthikonda R N.; Schmitt, S M. 1994 U.S. Pat. No.    5,292,879 A

(S)-Boc-2-[5-(Toluene-4-sulfonyl)-tetrazol-1-ylmethyl]-pyrrolidine) (2)

A sealed tube was charged with p-toluenesulfonyl cyanide (5.0 g, 22.1mmol) and (S)-Boc-2-azido methyl pyrrolidine (1) [Black, J.; Brown, A.D.; Erizabet C. L.; Smith, J. D,; Mckelloy, A. B. 2000 JP2000063380/](4.0 g, 22.1 mmol). The mixture was stirred at 80° C. for 40 hrs. Thecrude product was purified on flash silica gel column with(Hexane:EtOAc=1:1) to furnish 2 (7.6 g, 84%) as colorless foam. [α]_(D)−11.7 (c 0.64, CHCl₃). ¹H NMR (400 MHz, CD₃OD, −20° C., rotamers 6:4): δ1.20 & 1.35 (s, 9H), 1.83 & 2.01 (m, 4H), 2.50 (s, 3H) 3.40 & 3.45 (m,2H), 4.40 & 4.43 (m, 1H), 4.71 & 4.82 (m, 2H), 7.50 & 7.70 (d, J=8 Hz,2H), 8.00 & 8.40 (d, J=8 Hz, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 22.0, 23.7,28.4, 46.5, 52.1, 56.1, 76.9, 80.6, 129.4, 134.4, 147.4, 154.4, 154.8,155.5; IR (film): 2976, 1693, 1392, 815, 704 cm⁻¹; MS: (ESI) [M+1]⁺408.2.

(S)-Boc-2-(5-Phenylsulfanyl-tetrazol-1′-ylmethyl)-pyrrolidine (3a)

The mixture of 2 (6.0 g, 14.7 mmol), phenylthiol (6.5 g, 58.8 mmol) andK₂CO₃ (4.5 g, 41.1 mmol) in CH₃CN (74 ml) was stirred for 2 days at roomtemperature. The reaction mixture was filtered through a pad of Celiteand then concentrated in vacuo. The crude was purified by silica gelcolumn with (Hexane:EtOAc=1:1) as an eluant to give 3 (5.2 g, 97%) as ancolorless oil. [α]_(D) −29.2 (c 1.34, CHCl₃). ¹H NMR (400 MHz, CD₃OD,−20° C., rotamers 1:1): δ 1.30 & 1.42 (s, 9H), 1.73 & 2.01 (m, 4H) 3.40& 3.45 (m, 13.2 Hz, 2H), 4.23 & 4.25 (m, 1H), 4.38 & 4.58 (m, 2H), 7.42& 7.46 (m, 3H) 7.56 & 7.60 (m, 2H); ¹³C NMR (CDCl₃, 75 MHz): δ 21.2,23.6, 28.5, 46.5, 49.7, 50.4, 56.7, 60.5, 80.7, 128.1, 129.9, 133.1,153.5, 154.9; IR (film): 2975, 1693, 1391, 1167, 688 cm⁻¹; MS: (ESI)[M+1]⁺ 361.2

(S)-2-(5-phenylsulfanyl-tetrazol-1-ylmethyl)-pyrrolidine (4a)

3a (5.0 g, 13.8 mmol) was treated with 50% TFA in CH₂Cl₂ (69 ml) for 15min at room temperature. The mixture was sequestered with sat.NaHCO₃ andextracted with CH₂Cl₂ (3×20 ml). The combined extraction were dried overNa₂SO₄, filtered and concentrated. The crude was purified over flashsilica gel column with (CH₂Cl₂:MeOH:NH₄OH=20:0:9:1) as an eluant tofurnish 4a (2.5 g, 69%). [α]_(D) +9.9 (c 1.14, CHCl₃). ¹H NMR (400 MHz,CDCl₃): δ 1.45 (m, J=2, 8.4 Hz, 1H), 1.80 (m, 2H), 1.95 (m, 1H), 2.5(bs, 1H), 2.99 (t, J=1.6 Hz, 2H), 3.60 (m, 1H), 4.20 (dd, J=8.0, 13.0Hz, 1H), 4.30 (dd, J=8.0, 13.0 Hz 1H), 7.40 (m, 3H), 7.58 (m, 2H); ¹³CNMR (75 MHz, CDCl₃): δ 23.5, 28.6, 46.1, 47.8, 58.2, 127.2, 130.0,130.2, 133.1, 153.4; IR (film): 3350, 2961, 2871, 1442, 1389, 746, 688cm⁻¹; MS: (ESI) [M+1]⁺ 262.2.

{1-[2-(5-Phenylsulfanyl-tetrazol-1-ylmethyl)-pyrrolidine-1-carbonyl]-2-ynyloxy-propyl}-carbamicacid allyl ester (5a)

To a cold solution of 4a (2.5 g, 9.5 mmol) and 9 (2.7 g, 11.4 mmol) inDMF (48 ml) were added DIPEA (3.3 ml, 19.0 mmol) and HATU (5.4 g, 14.2mmol). After stirring for 30 min at 0° C., the reaction mixture wasdiluted with Et₂O. To the mixture was added sat.NaHCO₃ and thenseparated. The aqueous phase was extracted with Et₂O (3×30 ml). Thecombined extraction were washed with 5% HCl (25 ml), H₂O (25 ml),sat.NaHCO₃ (20 ml), and Brine (30 ml), and finally dried over MgSO₄,filtered and concentrated in vacuo. The crude was purified by silica gelcolumn with (hexane:EtOAc=1:1) to afford 5a (3.2 g, 70%) as an oil.[α]_(D) −12.2 (c 1.30, CHCl₃) ¹H NMR (400 MHz, CDCl₃): δ 1.22 (d, J=6Hz, 3H), 1.75-1.90 (m, 4H), 2.50 (t, J=2.4 Hz, 1H), 3.70 (m, 2H) 4.00(m, 1H), 4.20 (dq, J=5.6, 18.4 Hz, 2H), 4.31 (m, 2H), 4.50 (m, 1H), 4.55(m, 2H), 4.62 (dd, J=4.2 Hz, 1H), 5.20 (d, J=9.6 Hz, 1H), 5.23 (d,J=10.2 Hz, 1H), 5.50 (d, J=8 Hz, 1H), 5.90 (m, 1H), 7.40 (m, 3H), 7.60(m, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 16.0, 24.3, 27.5, 47.9, 48.5, 56.5,56.7, 66.1, 74.2, 74.9, 75.0, 79.8, 117.9, 127.6, 129.9, 130.0, 132.7,133.3, 153.5, 156.4, 169.8; IR (film): 3292, 2979, 1715, 1644, 1513,1442, 1073, 750, 688 cm⁻¹; MS: (ESI) [M+1]⁺ 485.2.

{1-[2-(5-Phenylsulfanyl-tetrazol-1-ylmethyl)-pyrrolidine-1-carbonyl]-2-ynyloxy-propyl}-ethyl)-carbamicacid 9H-fluoren-9-ylmethyl ester (6a)

To a solution of Fmoc-N(Me) L-Ala (6 g, 18.5 mmol) and HOBt (2.5 g, 18.5mmol) in CH₂Cl₂ (60 ml) was added EDCI (3.5 g, 18.5 mmol) at 0° C.stirred for 1 hr and an additional hour at room temperature. To thereaction mixture was added Pd (PPh₃)₄ (3.6 g, 3.0 mmol), a solution of5a (3 g, 6.2 mmol) in CH₂Cl₂ (30 ml) and DABCO (3.7 g, 31 mmol) at roomtemperature followed by stirred for 15 min. The reaction mixture wasconcentrated in vacuo and purified by silica gel column with(hexane:EtOAc=1:1) to furnish 6a (3.8 g, 89%) as pale yellow foam.[α]_(D) −31.9 (c 1.56, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 1.20 (d, J=3.6Hz, 3H), 1.41 (d, J=4.2 Hz, 3H), 1.75-1.98 (bm, 4H), 2.50 (bs, 1H), 2.95(s, 3H), 3.60 (bm, 1H), 3.70 (bm, 1H), 4.01 (bm, 1H), 4.10 (dq J=5.6 Hz,18.1 Hz, 2H), 4.20 (bm, 1H), 4.25 (bm, 1H), 4.28 (bm, 2H) 4.32 (bm, 1H),4.40 (bm, 2H), 4.59 (dd, J=4.2, 8.0 Hz, 1H), 6.81 (bd, 1H), 7.28 (bm,2H), 7.34 (bm, 5H), 7.52 (br m, 4H), 7.72 (d, J=7.6 Hz, m, 2H); ¹³C NMR(75 MHz, CDCl₃): δ 16.1, 24.3, 27.5, 47.4, 48.5, 55.3, 56.4, 68.2, 74.0,75.0, 79.8, 120.1, 125.2, 125.3, 127.2, 127.6, 127.9, 128.6, 129.9,130.1, 132.2, 132.4, 133.3, 141.5, 153.5, 169.3, 171.4; IR (film): 3297,2978, 1693, 1650, 1442, 1400, 1312, 1157, 10953, 758, 742 cm⁻¹; MS:(ESI) [M+1]⁺ 708.2

{1-[2-Bis-(5-Phenylsulfanyl-tetrazol-1-ylmethyl)-pyrrolidine-1-carbonyl]-2-ynyloxy-propyl}-ethyl)-carbamicacid 9H-fluoren-9-ylmethyl ester (7a)

A mixture of 6a (3.5 g, 4.9 mmol) and Cu(OAc)₂ (6.2 g, 34.3 mmol) inCH₃CN was refluxed for 30 min. The organic solvent was stripped off andthe resultant residue redissolved in CH₂Cl₂ and the Cu(II) salts wereremoved by filtering over a short pad of silica gel eluting with(CH₂Cl₂:MeOH=9:1) to give material, which was completely dried off tofurnish crude of 7a (3.3 g, 94%) as a mixture of desired product (major)and partially deprotected material. A small portion of the crude waspurified for spectral analysis. [α]_(D) −13.8 (c 1.41, CHCl₃). ¹H NMR(400 MHz, CDCl₃): δ 1.14 (d, J=6 Hz, 6H), 1.34 (d, J=7.2 Hz, 6H),1.74-1.98 (bm, 8H), 2.84 (s, 6H), 3.60 (bm, 4H), 3.87 (bm, 2H), 4.09(ABq, J=16.8 Hz, 4H), 4.20 (bm, 2H), 4.24 (m, 2H), 4.31 (bm, 4H), 4.36(m, 4H), 4.60 (dd, J=4.2 Hz, 2H), 4.69 (dd, J=4, 8.8 Hz, 2H), 6.80 (bm,2H), 7.28 (bm, 4H), 7.34 (bm, 10H), 7.52 (bm, 8H), 7.72 (d, J=7.6 Hz, m,4H); ¹³C NMR (75 MHz, CDCl₃): δ 16.3, 24.3, 27.5, 47.4, 48.5, 55.3,56.4, 68.2, 74.0, 75.0, 79.8, 120.1, 125.2, 125.3, 127.2, 127.6, 127.9,128.6, 129.9, 130.1, 132.2, 132.4, 133.3, 141.5, 153.5, 169.3, 171.4; IR(film): 2920, 2850, 1687, 1643, 1441, 1311, 1155, 1083, 741 cm⁻¹; MS:(ESI) [M+1]⁺ 1412.2

Bis-2-Methylamino-N-(1-{1-[2-(5-phenylsulfanyl-tetrazol-1-ylmethyl)-pyrrolidine-1-carbonyl]-2-ynyloxy-propyl}-propionamide(8a)

A solution of 7a (3 g, 2.1 mmol) was treated with 20% CH₂Cl₂-piperidine(21 ml) for 5 min. The mixture was concentrated in vacuo and thenpurified by silica gel column with (CH₂C12:MeOH:NH₄OH=8:2:0.5) tofurnish 8a (1.4 g 70%) as gummy foam. [α]_(D) −7.4 (c 0.23, CHCl₃). ¹HNMR (400 MHz, CDCl₃): δ 1.19 (d, J=6.4 Hz, 6H), 1.28 (d, J=7.2 Hz 6H),1.78 (m, 4H), 1.90 (m, 4H), 2.50 (s, 6H), 3.05 (q, J=7.2 Hz, 2H), 3.60(bm, 2H), 3.70 (m, 2H), 4.01 (m, 2H), 4.20 (ABq, J=18.4 Hz, 4H), 4.43(dd, J=4.0, 13.6 Hz, 2H), 4.50 (m, 2H), 4.70 (dd, J=4, 13.6 Hz, 2H),4.75 (dd, J=4, 8.8 Hz, 2H), 7.40 (m, 6H), 7.60 (m, 4H), 7.82 (d, J=8.4Hz, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 16.2, 19.6, 22.8, 24.4, 27.5, 31.8,35.2, 47.8, 48.5, 54.6, 56.3, 56.9, 60.4, 70.4, 74.4, 76.1, 76.8, 127.7,129.9, 130.0, 133.3, 153.6, 169.5, 175.4; IR (film): 3339, 2975, 1644,1513, 1428, 1086, 751, 667 cm⁻¹; MS: (ESI) [M+1]⁺ 969.4.

2-Methylamino-N-{2-(6-{1-methyl-2-(2-methylamino-propionylamino)-3-oxo-3-[2-(5-phenoxy-tetrazol-1-ylmethyl)-pyrrolidin-1-yl]-propoxy}-hexa-2,4-diynyloxy)-1-[2-(5-phenoxy-tetrazol-1-ylmethyl)-pyrrolidine-1-carbonyl]-propyl}-propionamide(8b)

8b was synthesized according to scheme 2.

¹H NMR (400 MHz, CDCl₃): δ 1.20 (d, J=6.4 Hz, 6H), 1.29 (d, J=6.8 Hz,6H), 1.65-2.15 (m, 8H), 2.50 (s, 6H), 3.05 (q, J=6.8 Hz, 2H), 3.59 (m,2H), 3.75 (m, 2H), 4.03 (m, 2H), 4.20 (ABq, J=11.6, 16.4 Hz, 4H), 4.39(m, 2H), 4.60 (m 4H), 4.67 (dd, J=4.4 Hz, 8.4 Hz, 2H), 7.20 (m, 2H),7.40 (m, 8H), 7.82 (d, J=8.4 Hz, 2H); MS: (ESI) [M+1]⁺ 937.5

N-(1-[2-(5-benzyloxy-tetrazol-1-ylmethyl)-pyrrolidine-1-carbonyl]-2-{6-[3-[2-(5-benzyloxy-tetrazol-1-ylmethyl)-pyrrolidin-1-yl]-1-methyl-2-(2-methylamino-propionylamino)-3-oxo-propoxy]-hexa-2,4-diynyloxy}-propyl)-2-methylamino-propionamide(8c)

8c was prepared according to scheme 3.

¹H NMR (400 MHz CDCl₃): δ 1.20 (d, J=6.4 Hz, 6H), 1.29 (d, J=6.8 Hz,6H), 1.60 (m, 4H), 1.81 (m, 4H), 2.50 (s, 6H), 3.05 (q, J=6.8 Hz, 2H),3.51 (m, 4H), 3.68 (m, 4H), 4.03 (m, 2H), 4.16 (m, 6H), 4.36 (dd, J=4.4,14.4 Hz 2H), 4.47 (m, 2H), 4.67 (dd, J=4.4, 8.4 Hz, 2H), 5.52 (ABq,J=11.6, 16.4 Hz, 4H), 7.37 (m, 6H), 7.48 (m, 4H), 7.82 (d, J=8.4 Hz,2H); MS: (ESI) [M/2]⁺ 482.4.

N-(1-[2-(5-benzylamino-tetrazol-1-ylmethyl)-pyrrolidine-1-carbonyl]-2-{6-[3-[2-(5-benzylamino-tetrazol-1-ylmethyl)-pyrrolidin-1-yl]-1-methyl-2-(2-methylamino-propionylamino)-3-oxo-propoxy]-hexa-2,4-diynyloxy}-propyl)-2-methylamino-propionamide(8d)

8d was synthesized according to scheme 4

¹H NMR (400 MHz, CDCl₃): δ 1.15 (D, J=6.4 Hz, 6H), 1.29 (d, J=6.8 Hz,6H), 1.85-2.20 (m, 8H), 2.50 (s, 6H), 3.05 (q, J=6.8 Hz, 2H), 3.60 (bm,2H), 3.79 (m, 2H), 3.96-4.40 (m, 14H), 4.64 (dd, J=8.8, 14.8 Hz 2H),4.73 (dd, J=3.2, 8.8 Hz, 2H), 6.81 (t, J=6.0 Hz, 2H), 7.22 (m, 6H), 7.39(m, 4H), 7.84 (d, J=8.8 Hz, 2H); MS: (ESI) [M/2+Na]⁺ 485.4.

Bis-N-(1-{2-[5-Benzylmethylamino]-tetrazol-1-ylmethyl}-pyrrolidine-1-carbonyl)-2-prop-2-ynyloxy-propyl)-2-methylamino-propioamide(8e)

8e was synthesized according to scheme 5.

¹H NMR (400 MHz, CDCl₃): δ 1.15 (D, J=6.4 Hz, 6H), 1.29 (d, J=6.8 Hz,6H), 1.79-2.15 (m, 8H), 2.50 (s, 6H), 3.05 (q, J=6.8 Hz, 2H), 3.15 (s,6H), 3.60 (bm, 4H), 3.99 (m, 2H), 4.10 (m, 6H), 4.41 (m, 2H, 4.51 (m,4H), 4.71 (m 4H), 7.25 (m, 10H), 7.80 (d, J=8.8 Hz, 2H); MS: (ESI)[M+Na]⁺ 1013.5.

(2S,4R)-2-tert-Butyl-4-(3-chloro-benzyl)oxazolidine-3,4-dicarboxylicacid 3-tert-butyl ester 4-methyl ester (11)

Under N₂, to a solution of 10 [1. Cagnon, J.; Bideau, F.;Marchand-Brynaert, J.; Ghosez, L. Tetrahedron Lett., 1997, 38, 2291; 2.Seebach, D.; Aebi, J. D. Tetrahedron Lett. 1984, 25(24), 2545-2548; 3.Seebach, V. D.; Aebi, J.; Gander-Coquoz, M.; Naef, R., Helvetica ChimicaActa, 1987, 70, 1194-1216] (1.0 mmol) and 3-chloro benzyl bromide (1.2mmol) in THF-HMPA (4:1, 10 mL) was added dropwise LHMDS (1.2 mmol) inTHF at −78° C. over 30 min. After additional stirring for 30 min at sametemperature (−78° C.), the reaction mixture was quenched with sat.NH₄Cl(5 mL), and allowed to warm up to room temperature. The separated waterphase was extracted with Et₂O (3×20 mL). The combined extraction werewashed with H₂O (20 mL) and Brine (20 mL), dried over MgSO₄, filteredand concentrated in vacuo to give the residue. The crude of 11 was usedfor next step without further purification. A small amount of crude wassubmitted to silica gel column chromatography with Hexan:EtOAc=20:1 aselute and taken for spectral data. [α]_(D) −67.3 (c 1.02, CHCl₃); ¹H NMR(CDCl₃, 300 MHz) δ 2.74 (d, J=13.2 Hz, 1H), 3.05 (d, J=13.2 Hz, 1H),3.57 (dd, J=1.5, 10.8 Hz, 1H), 3.73 (s, 3H), 3.87 (d, J=10.8 Hz, 1H),7.00 (m, 1H), 7.12 (m, 1H), 7.20-7.25 (m, 2H); ¹³C NMR (CDCl₃, 75 MHz) δ26.8, 28.3, 38.5, 39.5, 52.6, 69.4, 73.8, 81.5, 97.6, 127.1, 128.4,129.5, 130.4, 134.0, 138.1, 153.0, 172.0; IR (film): 2976, 1743, 1711,1359, 1136 cm⁻¹; Mass (ESI) 412.19 ([MH]⁺).

(R)-2-Amino-2-(3-chloro-benzyl)-3-hydroxy-propionic acid methyl ester(12)

Under N₂, to a solution of crude of 11 (1 mmol) in dried MeOH (5 mL) wasadded dropwise conc.HCl (2 mL) over 1 hr then stirred for overnight.After concentration, the residue was basified with NaHCO₃ at 0° C. Themixture was extracted with CH₂Cl₂ (5×20 mL). The combined extractionwere dried over MgSO₄, filtered and concentrated. The crude wassubmitted to silica gel column chromatography(CH₂Cl₂:MeOH:NH₄OH=500:9:1-100:9:1) to give 12 (95%, from 10). [α]_(D)−3.6 (c 1.45, CHCl₃); ¹H NMR (CDCl₃, 400 MHz) δ 2.75 (d, J=13 Hz, 1H),3.05 (d, J=13 Hz, 1H), 3.56 (d, J=10 Hz, 1H), 3.73 (s, 3H), 3.87 (d,J=10 Hz, 1H), 7.02 (m, 1H), 7.13 (m, 1H), 7.22-7.27 (m, 2H). ¹³C NMR(CDCl₃, 75 MHz) δ 41.5, 52.4, 63.6, 67.7, 127.4, 128.0, 129.8, 129.9,134.3, 137.5, 175.3; IR (film): 3359, 3300, 3180, 1736, 1083 cm⁻¹; Mass(ESI) 244.02 ([MH]⁺).

(2S,1′R)1-Fmoc-2-[2′-(3″-Chloro-phenyl)-1′-hydroxymethyl-1′-methoxycarbonyl-ethylcarbamoyl]-pyrrolidine(13)

Under N₂, to a solution of Fmoc-Pro-OH (1.0 mmol) and amino alcohol 12(1.0 mmol) in CH₃CN (10 mL) was added DMT-MM (1 mmol) then stirred for 3hr. The reaction mixture was concentrated to half amount. The residuewas diluted with Et₂O (30 mL), washed with 5% HCl (20 mL), H₂O (20 mL),5% NaOH (20 mL), H₂O (20 mL) and Brine (20 mL), dried over MgSO₄,filtered and concentrated. The crude was used for next step withoutfurther purification. [α]_(D) −41.8 (c 1.75, CHCl₃); ¹H (CDCl₃, 400 MHz)δ 1.82-1.94 (m, 2H), 2.06-2.30 (m, 2H), 3.02 (d, J=13.4 Hz, 1H),3.44-3.58 (m, 3H), 3.64 (d, J=13.4 Hz, 1H), 3.77 (s, 3H), 3.84 (t,J=10.1 Hz, 1H), 4.18-4.49 (m, 5H), 6.89 (d, J=6.6 Hz, 1H), 7.05 (s, 2H),7.16 (m, 2H), 7.30 (t, J=7.3 Hz, 2H), 7.39 (t, J=7.3 Hz, 2H), 7.56 (d,J=7.6 Hz, 2H), 7.75 (d, J=7.6 Hz, 2H). ¹³C (CDCl₃, 75 MHz) δ 24.4, 29.3,36.2, 46.9, 52.9, 62.0, 64.0, 67.6, 68.0, 119.9, 124.9, 125.0, 127.0,127.5, 127.7, 129.4, 129.7, 133.9, 137.5, 141.1, 141.2, 143.4, 143.8,156.1, 171.4, 172.0; IR (film): 3392, 1741, 1682, 1418, 758 cm⁻¹; Mass(ESI) 585.10 ([MNa]⁺).

(4R,2′S)-4-(3″-Chloro-benzyl)-2-[1′-Fmoc-pyrrolidin-2′-yl]-4,5-dihydro-oxazole-4-carboxylicacid methyl ester (14)

Under N₂, to a cold (−78° C.) solution of 13 (1.0 mmol) in CH₂Cl₂ (5 mL)was added DAST (1.2 mmol) dropwise within 10 min at −78° C. then stirredfor 1 hr at −78° C. After addition of K₂CO₃ (1.5 mmol), the reactionmixture was allowed to warm up to room temperature. The reaction mixturewas diluted with sat.NaHCO₃ (10 mL) then extracted with Et₂O (3×20 mL).The combined extractions were washed with H₂O (10 mL) and Brine (10 mL),filtered and concentrated in vacuo. The residue was purified by silicagel column chromatography (Hexane:EtOAc=2:1) to afford product 14 (72%,from 14) as white form. [α]_(D) −61.6 (c 1.90, CHCl₃); ¹H NMR (CDCl₃,300 MHz, mixture of rotamer) δ 1.77-2.21 (m, 4H), 3.07 and 3.14 (ABq,J=13.7/13.7 Hz, 2H), 3.44-3.72 (m, 2H), 3.56 and 3.70 (s, 3H), 4.03-4.62(m, 6H), 6.98-7.24 (m, 4H), 7.30 (t, J=7.3 Hz, 2H), 7.39 (t, J=7.3 Hz,2H), 7.61 (m, 2H), 7.75 (dd, J=5.1, 7.3 Hz, 2H); ¹³C NMR (CDCl₃, 75 MHz,mixture of rotamer) δ 23.4 and 24.4, 30.3 and 31.4, 42.6 and 43.0, 46.6and 47.0, 47.1 and 47.3, 52.6 and 52.6, 54.3 and 54.8, 67.3 and 67.5,73.3 and 73.3, 77.8 and 78.0, 119.9, 125.0 and 125.1, 125.1 and 125.2,127.0 and 127.0, 127.2 and 127.3, 127.6, 128.5, 129.5 and 129.5, 130.4and 130.5, 133.9 and 134.0, 136.9 and 137.2, 141.1 and 141.2, 141.2,141.2, 143.8 and 143.9, 144.0 and 144.2, 154.4 and 154.6, 169.2 and169.6, 172.7 and 172.8; IR (film): 2952, 1735, 1705, 1417 cm⁻¹; Mass(ESI) 567.15 ([MNa]⁺).

(2S,3S)-1-Alloc-3-Methyl-aziridine-2-carboxylic acid methyl ester

Under N₂, to a solution of known compound(2S,3S)-3-methyl-1-trityl-aziridine-2-carboxylic acid methyl ester [1.Mckeever, B.; Pattenden, G. Tetrahedron, 2003, 59, 2713-2727; 2. Wipf,P.; Uto, Y., J. Org. Chem., 2000, 65, 1037-1049] (20 g, 56 mmol) inCH₂Cl₂ (100 mL) and MeOH (2.3 mL) was added TFA (8.6 mL, 112 mmol) at 0°C. then stirred for 1 hr. Evaporation of the solvent in vacuo gave asolid residue which was dissolved in Et₂O. The solvent was evaporated,and the Et₂O treatment was repeated two additional times to ensurecomplete removal of TFA. The residue was dissolved in Et₂O and thenextracted with H₂O (5×30 mL). To the combined H₂O phase was addedportionwise NaHCO₃ carefully at 0° C. followed by EtOAc (56 mL) anddropwise Allyl chloroformate (8.9 mL, 84 mmol) at 0° C. then stirred for16 hr at room temperature. The reaction mixture was extracted with EtOAc(3×100 mL). The combined extraction were washed with H₂O (50 mL) andBrine (50 mL), dried over MgSO₄, filtered and concentrated. The crudewas purified by silica gel column chromatography with Hexane:EtOAc=10:1as eluant to give product (59%). [α]_(D) −81.7 (c 1.02, CHCl₃); ¹H NMR(CDCl₃, 400 MHz) δ 1.36 (d, J=5.7 Hz, 3H), 2.83 (dq, J=5.7, 6.7 Hz, 1H),3.19 (d, J=6.7 Hz, 1H), 3.79 (s, 3H), 4.61 (m, 2H), 5.26 (dd, J=1.2,10.4 Hz, 1H), 5.33 (dd, J=1.5, 17.2 Hz, 1H), 5.91 (ddt, J=5.7, 10.4,17.2 Hz, 1H); ¹³C NMR (CDCl₃, 75 MHz) δ 12.8, 38.8, 39.7, 52.3, 67.3,118.8, 131.5, 161.3, 167.5; IR (film): 1733, 1296, 1282, 1204 cm⁻¹; Mass(ESI) 200.10 ([MH]⁺).

(2S,3R)-2-Alloc-amino-3-prop-2′-ynyloxy-butyric acid methyl ester

Under N₂, to a solution of above product (3.2 g, 16 mmol) in propargylalcohol (186 mL) was added BF₃.Et₂O (4.1 mL, 32 mmol) at roomtemperature then stirred for 1 hr. The reaction mixture was concentratedin vacuo. The residue was dissolved in H₂O (50 mL) and then extractedwith Et₂O (3×40 mL). The combined extraction were washed with H₂O (30mL) and Brine (30 mL), dried over MgSO₄, filtered and concentrated. Thecrude was purified by silica gel column chromatography withHexane:AcOEt=10:1 as eluant to give propargyl Threonine derivative(95%). [α]_(D) 6.9 (c 1.53, CHCl₃); ¹H NMR (CDCl₃, 400 MHz) δ 1.24 (d,J=6.2 Hz, 3H), 2.41 (t, J=2.4 Hz, 1H), 3.77 (s, 3H), 4.12 (qd, J=2.4,16.1 Hz, 2H), 4.31 (m, 1H), 4.36 (dd, J=2.2, 9.5 Hz, 1H), 4.60 (d, J=5.5Hz, 2H), 5.22 (dd, J=1.1, 10.4 Hz, 1H), 5.33 (dd, J=1.5, 17.2 Hz, 1H),5.43 (br d, J=9.3 Hz, 1H), 5.93 (ddt, J=5.5, 10.8, 17.2 Hz, 1H); ¹³C NMR(CDCl₃, 75 MHz) δ 15.9, 52.4, 56.0, 58.5, 65.9, 73.8, 74.4, 79.2, 117.7,132.6, 156.5, 171.0; IR (film): 3292, 1750, 1724, 1517, 1212, 1075 cm⁻¹;Mass (ESI) 256.17 ([MH]⁺)

(2S,3R)-2-Alloc-lamino-3-prop-2′-ynyloxy-butyric acid (9)

Under N₂, a solution of above material (1 g, 3.9 mmol) and 0.26N HCl (15mL) in AcOH (45 mL) was refluxed for 20 hr. The reaction mixture wasconcentrated. The crude was passed short column chromatography withHexane:EtOAc=1:1 as eluant to give 9 (95%). [α]_(D) −1.6 (c 0.99,CHCl₃); ¹H NMR (CDCl₃, 400 MHz) δ 1.27 (d, J=6.4 Hz, 3H), 2.45 (t, J=2.2Hz, 1H), 4.17 (qd, J=2.2, 15.9 Hz, 2H), 4.34 (m, 1H), 4.41 (d, J=9.2 Hz,1H), 4.60 (d, J=5.5 Hz, 2H), 5.23 (d, J=10.7 Hz, 1H), 5.32 (d, J=17.2Hz, 1H), 5.47 (br d, J=9.2 Hz, 1H), 5.92 (ddt, J=5.5, 10.7, 17.2 Hz,1H); ¹³C NMR (CDCl₃, 75 MHz) δ 16.3, 56.5, 58.7, 66.3, 74.2, 75.2, 79.2,118.1, 132.7, 156.9, 175.8; IR (film): 3293, 1717, 1522, 1076 cm⁻¹; Mass(ESI) 242.04 ([MH]⁺).

(4R,2′S)-2-[1′-Alloc-Thr(propargyl)-pyrrolidin-2′-yl]-4-(3″-chloro-benzyl)-4,5-dihydro-oxazole-4-carboxylicacid methyl ester (15)

14 (634 mg, 1.16 mmol) was treated with 20% piperidine-CH₂Cl₂ (10 mL).The reaction mixture was concentrated. The crude was through shortcolumn chromatography with CH₂Cl₂:MeOH:NH₄OH=200:9:1 as elute. Theresidue was dissolved in DMF and followed by adding 9 (309 mg, 1.28mmol), DIPEA (404 μL, 2.32 mmol) and HATU (487 mg, 1.28 mmol) at 0° C.then stirred for 30 min. The reaction mixture was diluted with Et₂O andsat.NaHCO₃ was added. The aqueous phase was extracted with Et₂O. Thecombined extraction were washed with H₂O and Brine, dried over MgSO₄,filtered and concentrated. The crude was purified by silica gel columnchromatography with Hexane:EtOAc=1:1 as eluant to give 15 (55%). ¹H NMR(CDCl₃, 400 MHz, mixture of rotamer) δ 1.20 and 1.24 (d, J=6.2/6.2 Hz,3H), 1.83-2.24 (m, 4H), 2.40 and 2.43 (t, J=2.4/2.4 Hz, 1H), 3.11 and3.27 (ABq, J=13.7/13.9 Hz, 2H), 3.72-3.75 (s, 3H), 3.50-4.97 (m, 13H),5.20-5.30 (m, 2H), 5.68 and 5.78 (d, J=8.2/8.4 Hz, 1H), 5.90 (m, 1H),7.08 (m, 1H), 7.17-7.25 (m, 3H); Mass (ESI) 546.15 ([MH]⁺), 568.10([MNa]⁺)

(4R,2′S)-2-[1′-Fmoc-Ala-Thr(propargyl)-pyrrolidin-2′-yl]-4-(3″-chloro-benzyl)-4,5-dihydro-oxazole-4-carboxylicacid methyl ester (16)

Under N₂, to a solution of Fmoc-Ala-OH (633 mg, 1.9 mmol) and HOBt (305mg, 1.9 mmol) in CH₂Cl₂ (40 mL) was added EDCI (369 mg, 1.9 mmol) at 0°C. and then stirred for 1 hr at 0° C. and additional 1 hr at roomtemperature. To the reaction mixture was added Pd(PPh₃)₄ (295 mg, 0.26mmol), a solution of 15 (350 mg, 0.64 mmol) and DABCO (359 mg, 3.2 mmol)and then stirred for 20 min. The reaction mixture was concentrated invacuo and then submitted to silica gel column chromatography withHexane:EtOAc=1:1˜1:9 as eluent to give 16 (99%). ¹H NMR (CDCl₃, 400 MHz,mixture of rotamer) δ 1.17 and 1.22 (d, J=6.4/6.4 Hz, 3H), 1.40 (br d,J=7.7 Hz, 3H), 1.81-2.16 (m, 4H), 2.31 and 2.38 (t, J=2.4/2.4 Hz, 1H),3.10 and 3.24 (ABq, J=13.9/13.9 Hz, 2H), 3.72 and 3.74 (s, 3H),3.70-5.00 (m, 9H), 5.36 (m, 1H), 6.75 (d, J=7.3 Hz, 1H), 7.05-7.70 (m,10H), 7.76 (d, J=7.5 Hz, 2H); Mass (ESI) 755.15 ([MH]⁺).

Compound 17a.

Under O₂, the mixture of 18 (34 mg, 0.045 mmol), CuI (8.6 mg, 0.045mmol) and pyridine (11 μL, 0.135 mmol) in THF (0.45 mL) was stirred for20 hrs. The reaction mixture was diluted with 10% glycine aq andextracted with CH₂Cl₂ (5×5 mL). The combined extraction were dried overMgSO₄, filtered and concentrated. The crude of 19 was treated with 20%piperidine-DMF for 10 min. The reaction mixture was concentrated andpurified by preparative TLC with CH₂Cl₂:MeOH:NH₄OH=100:9:1 as eluent togive 17a (46% from 18). ¹H NMR (CDCl₃, 400 MHz, 20 has rotamer, onlymajor rotamer was shown) δ 1.21 (d, J=6.2 Hz, 6H), 1.36 (d, J=7.0 Hz,6H), 1.80-2.12 (m, 8H), 3.11 (ABq, J=13.7 Hz, 4H), 3.48-3.96 (m), 4.15(d, J=9.2 Hz, 2H), 4.30 (q, J=16.5 Hz, 4H), 4.58 (d, J=9.2 Hz 2H),4.61-4.93 (m), 7.06-7.10 8 (m, 2H), 7.17-7.24 (m, 6H), 7.94 (d, J=8.4Hz, 2H); Mass (ESI) 532.38 ([[M⁺/2]H]⁺), 1063.61 ([MH]⁺).

N-Me dimer (R=Me, 17b) was synthesized by same procedure as abovedescribed for 17a.

¹H NMR (CDCl₃, 400 MHz, 17b has rotamer) δ 1.19 and 1.22 (d, J=6.2/6.4Hz, 6H), 1.29 and 1.31 (d, J=6.6/6.8 Hz, 6H), 1.80-2.12 (m, 8H), 2.40and 2.42 (s, 6H), 3.10 and 3.28 (ABq, J=13.6/13.8 Hz, 4H), 3.72 and 3.74(s, 6H), 3.71-3.96 (m, 6H), 4.14 and 4.28 (A of ABq, J=9.2/9.0 Hz, 2H),4.31 (ABq, J=16.3 Hz, 2H), 4.58 and 4.62 (B of ABq, J=9.2/9.0 Hz, 2H),4.66-4.94 (m, 2H), 7.06-7.10 (m, 2H), 7.18-7.25 (m, 6H), 7.80 (d, J=8.4Hz, 2H); Mass (ESI) 548.85 ([[M⁺/2]H]⁺), 1113.35 ([MNa]⁺).

[5-(3,5-Diiodo-phenylcarbamoyl)-pentyl]-carbamic acid2-trimethylsilanyl-ethyl ester (21)

3,5-Diiodoaniline [Dininno, F.; Guthikonda, R. N.; Schmitt, S. M. 1994U.S. Pat. No. 5,292,879 A] (1 g, 2.90 mmol),6-(2-Trimethylsilanyl-ethoxycarbonylamino)-hexanoic acid (1 g, 3.77mmol) in DMF (12 ml) were added DIPEA (0.9 ml, 5.8 mmol) and HATU (1.65g, 4.35 mmol). The mixture was stirred for overnight at roomtemperature. The reaction mixture was diluted with Et₂O. To the mixturewas added sat.NaHCO₃ and then separated. The aqueous phase was extractedwith Et₂O (3×40 ml). The combined extraction were washed with 5% HCl (30ml), H₂O (50 ml), sat.NaHCO₃ (30 ml), and Brine (30 ml), and finallydried over MgSO₄, filtered and concentrated in vacuo. The crude waspurified by silica gel column with (Hexane:EtOAc=6:4) to afford 21 (1 g,59%) as a colorless powder. ¹H NMR (300 MHz, (CD₃)₂SO): δ 0.09 (S, 9H),0.84 (t, J=2.1 Hz, 2H), 1.34 (m, 2H), 1.40 (m, 2H), 1.50 (m, 2H), 2.41(t, J=1.8 Hz, 2H), 2.47 (m, 2H), 2.89 (m, 2H), 4.01 (t, 2H), 6.92 (bt,1H), 7.68 (m, 1H), 7.99 (m, 2H); ¹³C NMR (75 MHz, (CD₃)₂SO): δ −0.72,18.0, 25.2, 26.4, 29.9, 36.9, 61.9, 96.4, 127.0, 139.0, 142.3, 156.9,172.2; IR (film): 3326, 2853, 2361, 1687, 1541, 1450, 1249, 837 cm⁻¹;MS: (ESI)) [M+Na]⁺ 624.9

6-[4-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-6-yl)-butyrylamino]-hexanoicacid(3,5-diiodo-phenyl)-amide (22a)

The above compound 21 (0.37 g, 0.61 mmol) was treated with 50%TFA-CH₂Cl₂ (4 ml) for 1 hr. The solvent was evaporated to furnish acrude, which was subjected for next step without any furtherpurification.

The salt (0.34 g, 0.59 mmol), (+)-Biotin (0.18 g, 0.76 mmol) in DMF (4ml) were added DIPEA (0.3 ml, 1.7 mmol) and HATU (0.33 g, 0.88 mmol).The mixture was stirred for overnight at room temperature. The reactionmixture was concentrated in vacuo, The crude was purified by silica gelcolumn with (MeOH:CHCl₃:NH₄OH=1:9:0.1) to afford 22a (0.3 g, 84%) asflakes. [α]_(D) +16 (c 1, DMF). ¹H NMR (400 MHz, (CD₃)₂SO): δ 1.25-1.60(m, 12H), 1.95 (t, J=1.8 Hz, 2H), 2.27 (m, 2H), 2.89 (m, 2H), 3.01 (m,2H), 3.15 (m, 4H), 3.61 (m, 2H), 4.15 (m, 1H), 4.25 (m, 1H), 6.40 (d,1H), 7.60 (s, 1H), 8.00 (s, 2H); ¹³C NMR (75 MHz, (CD₃)₂SO): 25.2, 26.0,26.6, 28.6, 28.8, 29.6, 35.8, 36.9, 38.7, 42.4, 54.0, 56.1, 59.7, 96.4,126.9, 139.0, 142.2; IR (film) 3296, 2930, 1675, 1571, 1201, 719 cm⁻¹;MS: (ESI) [M+Na]⁺ 707.

6-[4-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-6-yl)-butyrylamino]-hexanoicacid(3,5-bis-(3-{1-methyl-2-(1-methylamino-ethylamino)-3-oxo-3-[2-(5-phenylsulfanyl-tetrazol-1-ylmethyl)-pyrrolidin-1-yl]-propoxy}-prop-1-ynyl)-phenyl)-amide(24a)

22a (0.026 g, 0.039 mmol) and 23a (0.038 g, 0.078 mmol) in DMF wereadded PdCl₂(PPh₃)₂ (2.7 mg, 10 mol %), CuI (1.1 mg, 16 mol %) and Et₃N(27 μl, 0.19 mmol). The reaction mixture was stirred for 5 hrs at roomtemperature. Solvent was evaporated and the resultant mixture waspurified by preparative TLC using (CHCl₃:MeOH:NH₄OH=8:2:0.4) to furnishthe desired product 24a in 60% chemical yield. [α]_(D) +10 (c 1, CHCl₃).¹H NMR (400 MHz, CDCl₃): δ 1.09 (d, J=8.4 Hz, 6H), 1.11 (d, J=6.8 Hz,6H), 1.45-1.81 (m, 20H), 1.97 (t, J=7.6 Hz, 2H), 2.13 (dd, J=4.4, 12 Hz,2H), 2.21 (s, 6H), 2.53 (d, J=12.8 Hz, 1H), 2.89 (m, 5H), 3.40 (m, 2H),3.42-3.60 (m, 4H), 3.95 (m, 2H), 4.15 (m, 2H), 4.19 (ABq, J=19.0 Hz,4H), 4.37 (m, 2H), 4.45 (dd, J=4.8, 14 Hz, 2H), 4.57 (bd, 2H), 6.95 (s,1H), 7.22 (m, 6H), 7.38 (m, 4H), 7.78 (s, 2H); ¹³C NMR (75 MHz, CDCl₃):16.1, 19.6, 24.3, 24.9, 25.5, 26.2, 27.5, 27.7, 29.1, 35.2, 37.1, 39.2,40.9, 47.8, 48.8, 54.9, 55.7, 56.2, 56.8, 60.3, 61.9, 73.5, 84.8, 85.9,93.8, 122.1, 127.6, 128.8, 130.0, 133.5, 135.1, 139.8, 163.9, 169.7,173.4, 185.6; I.R: 3307, 2931, 1651, 1430, 1087, 667 cm⁻¹; MS: (ESI)[M+1]⁺ 1399.9.

Compound 33a.

33a was synthesized according to scheme 8.

¹H NMR (CDCl₃, 400 MHz) δ 1.18 (d, J=6.2 Hz, 3H), 1.23 (d, J=6.3 Hz,3H), 1.31 (d, J=7.0 Hz, 3H), 1.32 (d, J=7.0 Hz, 3H), 1.75-2.00 (m, 8H),2.40 (s, 3H), 2.43 (s, 3H), 3.07 (1, J=7.0 Hz, 1H), 3.40-3.78 (m, 7H),3.97 (dq, J=4.8, 6.2 Hz, 1H), 4.06 (dq, J=4.4, 6.2 Hz, 1H), 4.21-4.48(m, 5H), 4.68 (dd, J=4.0, 13.4 Hz 1H), 4.75 (dd, J=4.4, 8.8 Hz, 1H),7.40 (m, 3H), 7.62 (m, 2H), 7.81; (bd, J=8.6 Hz, 1H), 7.88 (bd, J=8.6Hz, 1H); (Mass (ESI) [MNa]⁺ 801.35.

6f-t is synthesized same procedure as 6a-e.

16c-t synthesized adopting similar procedure described for 16a,b.

31b-d is synthesized in a similar procedure described for 31a.

18 is synthesized in a similar procedure described for 8a-e.

19 is synthesized using similar procedure described for 24a.

60 is obtained by reacting 42a-t with propargyl dibromide in presence ofAg₂O in Et₂O.

43a-t is synthesized according to similar procedures as above describedfor 60 using treatment with propargyl bromide and Ag₂O in Et₂O, followedby standard work-up procedures

61 is synthesized according to similar procedures as described above for18 using Cu(OAc)₂ in CH₃CN.

62 may be synthesized according to similar procedures as described abovefor 18 using Cu(OAc)₂ in CH₃CN.

1. A method of promoting apoptosis of pathogenic cells, the methodcomprising the steps of contacting the cells with an effective amount ofa dimeric compound of formula II, and detecting a resultant increase inapoptosis of the cells,

wherein: R1 and R1′ are selected from hydrogen, optionally substitutedmethyl, and hydroxyl; R2 and R2′ are selected from optionallysubstituted methyl and optionally substituted ethyl; R3 and R3′ areselected from CH2, NH, O and S; R4 and R4′ are selected from CH and N;R5-R8, and R5′-R8′ are selected from hydrogen, optionally hetero-,optionally substituted alkyl, optionally hetero-, optionally substitutedalkenyl, optionally hetero-, optionally substituted alkynyl, optionallyhetero-, optionally substituted aryl, wherein optionally either R6 andR7 as well as R6′ and R7′ or R7 and R8 as well as R7′ and R8′ areconnected in 5- to 8-membered rings; and L is a contiguous chain ofbetween 2 and 200 atoms, having a MW between 20 and 2 KD that canincorporate substitution, heteroatoms, unsaturation and cyclic aromaticand heteroaromatic portions, covalently linking any of R2, R5, R6 or R7,with any of R2′, R5′, R6′ or R7′, or a pharmaceutically-acceptable saltthereof.
 2. The method of claim 1, wherein R1 and R1′ are selected fromhydrogen and methyl.
 3. The method of claim 1, wherein R2 and R2′ areselected from methyl and ethyl.
 4. The method of claim 1, wherein R3 andR3′ are NH.
 5. The method of claim 1, wherein R4 and R4′ are CH.
 6. Themethod of claim 1, wherein R5 and R5′ are C1-C3 alkyl.
 7. The method ofclaim 1, wherein R1 and R1′ are selected from hydrogen and methyl, R2and R2′ are selected from methyl and ethyl, R3 and R3′ are NH, R4 is CH,and R5 and R5′ are C1-C3 alkyl, and L covalently links R5, R6 or R7,with R5′, R6′ or R7′,
 8. The method of claim 1, wherein either R6 and R7as well as R6′ and R7′ or R7 and R8 as well as R7′ and R8′ are connectedin 5- to 8-membered rings.
 9. The method of claim 1, wherein R7 and R8are connected in a 5- or 6-membered ring.
 10. The method of claim 1,wherein R6 and R7 are connected in a 5- or 6-membered ring.
 11. Themethod of claim 1, wherein R6 and R7 are connected in a 5-membered ring,and L covalently links the ring with R2′, R5′, R6′ or R7′.
 12. Themethod of claim 1, wherein R8 comprises a 5- or 6-membered ring.
 13. Themethod of claim 1, wherein R8 comprises a 5-membered ring, comprising atleast one heteroatom, at least one substitution, and at least oneunsaturation.
 14. The method of claim 1, R1 and R2 and R1′ and R2′ areconnected in 5-membered rings.
 15. The method of claim 1, wherein L is acontiguous chain of between 4 and 100 atoms, and 40 and 1 kD.
 16. Themethod of claim 1, wherein L is an optionally hetero-, optionallysubstituted dialkynyl radical.
 17. The method of claim 1, wherein L issubstituted with a protonated or alkylated amine wherein the compound isa triamine.
 18. The method of claim 1, wherein the dimer is symmetricalabout the linker.
 19. The method of claim 1, wherein the dimer issymmetrical.
 20. The method of claim 1, wherein the cells are breastcancer cells.
 21. A method of treating a disease associated withundesirable caspase activity, the method comprising the steps ofadministering an effective dosage of a dimeric compound of formula II,and detecting a resultant decrease in pathology associated with thedisease, wherein the disease is an inflammatory or neoproliferativedisease,

wherein: R1 and R1′ are selected from hydrogen, optionally substitutedmethyl, and hydroxyl; R2 and R2′ are selected from optionallysubstituted methyl and optionally substituted ethyl; R3 and R3′ areselected from CH2, NH, O and S; R4 and R4′ are selected from CH and N;R5-R8, and R5′-R8′ are selected from hydrogen, optionally hetero-,optionally substituted alkyl, optionally hetero-, optionally substitutedalkenyl, optionally hetero-, optionally substituted alkynyl, optionallyhetero-, optionally substituted aryl, wherein optionally either R6 andR7 as well as R6′ and R7′ or R7 and R8 as well as R7′ and R8′ areconnected in 5- to 8-membered rings; and L is a contiguous chain ofbetween 2 and 200 atoms, having a MW between 20 and 2 KD that canincorporate substitution, heteroatoms, unsaturation and cyclic aromaticand heteroaromatic portions, covalently linking any of R2, R5, R6 or R7,with any of R2′, R5′, R6′ or R7′, or a pharmaceutically-acceptable saltthereof.
 22. A method of inhibiting a caspase, the method comprising thestep of: contacting a composition comprising a caspase with an effectiveamount of the compound of claim 1, and detecting a resultant decrease inactivity of the caspase, and

wherein: R1 and R1′ are selected from hydrogen, optionally substitutedmethyl, and hydroxyl; R2 and R2′ are selected from optionallysubstituted methyl and optionally substituted ethyl; R3 and R3′ areselected from CH2, NH, O and S; R4 and R4′ are selected from CH and N;R5-R8, and R5′-R8′ are selected from hydrogen, optionally hetero-,optionally substituted alkyl, optionally hetero-, optionally substitutedalkenyl, optionally hetero-, optionally substituted alkynyl, optionallyhetero-, optionally substituted aryl, wherein optionally either R6 andR7 as well as R6′ and R7′ or R7 and R8 as well as R7′ and R8′ areconnected in 5- to 8-membered rings; and L is a contiguous chain ofbetween 2 and 200 atoms, having a MW between 20 and 2 KD that canincorporate substitution, heteroatoms, unsaturation and cyclic aromaticand heteroaromatic portions, covalently linking any of R2, R5, R6 or R7,with any of R2′, R5′, R6′ or R7′, or a pharmaceutically-acceptable saltthereof.
 23. A WAP-RAS transgenic mouse, for use as an in vivo therapymodel for dimeric compounds of formula II.